Novel salts and pharmaceutical compositions thereof for the treatment of inflammatory disorders

ABSTRACT

The present invention discloses salts of a Compound 1: 
     
       
         
         
             
             
         
       
     
     useful in the prophylaxis and/or treatment of inflammatory conditions, autoimmune diseases, proliferative diseases, allergy, transplant rejection, diseases involving degradation and/or disruption of cartilage homeostasis, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims the priority of co-pending United KingdomApplication No. GB1402071.3 filed on Feb. 7, 2014, and the disclosure ofsaid application is incorporated herein by reference in its entirety.Applicants claim the benefits of said application under 35 U.S.C. §119.

FIELD OF THE INVENTION

The present invention relates to the salt and crystalline forms of acompound according to Formula I, useful in the prophylaxis and/ortreatment of inflammatory conditions, autoimmune diseases, proliferativediseases, allergy, transplant rejection, diseases involving degradationand/or disruption of cartilage homeostasis, congenital cartilagemalformations, and/or diseases associated with hypersecretion of IL6 orinterferons. In particular, the salt of the invention inhibits JAK, afamily of tyrosine kinases, and more particularly JAK1. The presentinvention also provides pharmaceutical compositions comprising the saltof the invention and methods for the prophylaxis and/or treatment ofdiseases including inflammatory conditions, autoimmune diseases,proliferative diseases, allergy, transplant rejection, diseasesinvolving degradation and/or disruption of cartilage homeostasis,congenital cartilage malformations, and/or diseases associated withhypersecretion of IL6 or interferons by administering a salt of theinvention according to Formula I.

BACKGROUND OF THE INVENTION

Current therapies for treating inflammatory conditions, autoimmunediseases, proliferative diseases, allergy, transplant rejection,diseases involving impairment of cartilage turnover, congenitalcartilage malformations, and/or diseases associated with hypersecretionof IL6 or interferons, in particular rheumatoid arthritis, are far fromsatisfactory and there remains a need to identify new therapeutic agentsthat may be of use in their treatment. These conditions are chronicconditions which require long term therapy, and repeated intake of thedrug. Long term treatment might be a heavy burden on the patient and thepractitioner alike, since the patient might be or become intolerant tothe drug, and furthermore high dosage, or high dosage frequency mayresult in uncomfortable side effects, and/or low patient compliance,where the patient may occasionally, deliberately or accidentally, miss adose. The impact of non-adherence varies across chronic illnesses, andranges from minimal to very significant (Ingersoll & Cohen, 2008).Therefore, there is a need to identify new agents to reinforce thearsenal of the practitioner, and compounds with low frequency dosageregimen to improve the life of the patients.

Janus kinases (JAKs) are cytoplasmic tyrosine kinases that transducecytokine signaling from membrane receptors to STAT transcriptionfactors. Four JAK family members are described, JAK1, JAK2, JAK3 andTYK2. Upon binding of the cytokine to its receptor, JAK family membersauto- and/or transphosphorylate each other, followed by phosphorylationof STATs that then migrate to the nucleus to modulate transcription.JAK-STAT intracellular signal transduction serves the interferons, mostinterleukins, as well as a variety of cytokines and endocrine factorssuch as EPO, TPO, GH, OSM, LIF, CNTF, GM-CSF and PRL (Vainchcnkcr, Dusa,& Constantincscu, 2008).

The combination of genetic models and small molecule JAK inhibitorresearch revealed the therapeutic potential of several JAKs.

JAK1 is a target in the immuno-inflammatory disease area. JAK1heterodimerizes with the other JAKs to transduce cytokine-drivenpro-inflammatory signaling. Therefore, inhibition of JAK1 is of interestfor immuno-inflammatory diseases with pathology-associated cytokinesthat use JAK1 signaling, such as IL-2, IL-6, IL-4, IL-5, IL-13, orIFNgamma, as well as for other diseases driven by JAK-mediated signaltransduction.

In the JAK family members' roles, some overlap exists, since mostsignaling pathways involve more than one JAK, however for some growthfactors such as erythropoietin and thrombopoietin, only JAK2 isinvolved.

JAK3 plays a major role in blocking immune function via transmission ofsignals generated by interleukin (IL)-2.

On the other hand, TYK2 would appear to work in combination with JAK2 inorder to transduce signaling of cytokines such as IL-12 and IL-23.

The role of JAK enzymes has been mostly studied using mice where each ofthe JAK family members has been deleted. JAK1 knockout mice exhibit aperinatal lethal phenotype and also have defective lymphoid developmentand function as a result of defective signaling by cytokines throughJAK1. JAK2 deficiency results in embryonic lethality at day 12 as aresult of a failure in definitive erythropoiesis. JAK3-deficient micehave severe combined immunodeficiency (SCID) phenotype but do not havenon-immune defects (Verstovsek, 2009).

As has been observed with pan JAK inhibitors, non-selective inhibitionmay be linked to side effects such as anemia, an increased rate ofinfections, lower neutrophil and lymphocyte counts, a decrease inhaemoglobin, and elevated cholesterol levels (Dolgin, 2011).

Therefore, the development of a selective JAK inhibitor would bebeneficial in order to minimize such side effects.

The degeneration of cartilage is the hallmark of various diseases, amongwhich rheumatoid arthritis and osteoarthritis are the most prominent.Rheumatoid arthritis (RA) is a chronic joint degenerative disease,characterized by inflammation and destruction of the joint structures.When the disease is untreated, it can lead to substantial disability andpain due to loss of joint function and result in shortenedlife-expectancy. The aim of a RA therapy, therefore, is not only to slowdown the disease but to attain remission in order to stop the jointdestruction and improve quality of life. Besides the severity of thedisease outcome, the high prevalence of RA (˜0.8% of adults are affectedworldwide) means a high socio-economic impact. (Smolen & Steiner, 2003)(O'Dell, 2004). JAK1 is implicated in intracellular signal transductionfor many cytokines and hormones. Pathologies associated with any ofthese cytokines and hormones can be ameliorated by JAK1 inhibitors.Hence, several allergy, inflammation and autoimmune disorders mightbenefit from treatment with compounds described in this inventionincluding rheumatoid arthritis, systemic lupus erythematosus, juvenileidiopathic arthritis, osteoarthritis, asthma, chronic obstructivepulmonary disease (COPD), tissue fibrosis, cosinophilic inflammation,cosophagitis, inflammatory bowel diseases (e.g. Crohn's disease,ulcerative colitis), transplant, graft-versus-host disease, psoriasis,myositis, psoriatic arthritis, ankylosing spondylitis, juvenileidiopathic arthritis, and multiple sclerosis. (Kopf, Bachmann, &Marsland, 2010)

Psoriasis is a disease that can affect the skin. The cause of psoriasisis not fully understood but it is believed that it is an immune mediatedrelated disease linked to the release of cytokines, in particular TNFα,which causes inflammation and rapid reproduction of the skin cells. Thishypothesis has been corroborated by the observation thatimmunosuppressant medication can clear psoriasis plaques. (Zenz, et al.,2005) Psoriasis can also cause inflammation of the joints, which isknown as psoriatic arthritis. Between 10-30% of all people withpsoriasis also have psoriatic arthritis. ((CHMP), 18 Nov. 2004) Becauseof its chronic recurrent nature, psoriasis is a challenge to treat. Ithas recently been demonstrated that inhibition of JAK could result insuccessful improvement of the psoriatic condition (Punwani, et al.,2012).

Inflammatory bowel disease (IBD) is a group of inflammatory conditionsof the colon and small intestine. The major types of IBD are Crohn'sdisease and ulcerative colitis. Recently, it has been found viagenome-wide association (GWAS) studies that T cell protein tyrosinephosphatise (TCPTP) is a JAK/STAT and growth factor receptor phosphatascthat has been linked to the pathogenesis of type 1 diabetes, rheumatoidarthritis, and Crohn's disease by GWAS. (Zikherman & Weiss, 2011)Therefore, inhibition of the JAK pathway might provide a way of treatingIBD.

JAK family members have been implicated in additional conditionsincluding myeloproliferative disorders (O'Sullivan, Liongue, Lewis,Stephenson, & Ward, 2007), where mutations in JAK2 have been identified.This indicates that inhibitors of JAK in particular JAK2 may also be ofuse in the treatment of myeloproliferative disorders. Additionally, theJAK family, in particular JAK1, JAK2 and JAK3, has been linked tocancers, in particular leukaemias (e.g. acute myeloid leukaemia(O'Sullivan, Liongue, Lewis, Stephenson, & Ward, 2007) (Xiang, et al.,2008) and acute lymphoblastic leukaemia (Mullighan, 2009)), cutaneousT-cell lymphoma (Zhang, 1996) or solid tumours e.g. uterineleiomyosarcoma (Constantinescu, Girardot, & Pecquet, 2007), prostatecancer (Tam, McGlynn, Traynor, Mukheijee, Bartlett, & Edwards, 2007) andbreast cancer (Berishaj, et al., 2007). These results indicate thatinhibitors of JAK, in particular of JAK1, may also have utility in thetreatment of cancers (leukaemias and solid tumours e.g. uterineleiomyosarcoma, prostate cancer, pancreatic cancers).

In addition, Castleman's disease, multiple myeloma, mesangialproliferative glomerulonephritis, psoriasis, and Kaposi's sarcoma arelikely due to hypersecretion of the cytokine IL-6, whose biologicaleffects are mediated by intracellular JAK-STAT signaling (Naka,Nishimoto, & Kishimoto, 2002). This result shows that inhibitors of JAK,may also find utility in the treatment of said diseases.

Thus, compounds which are potent inhibitors of JAK would offer thepotential for treating a wide variety of the disease and conditionsdescribed above.

The compound cyclopropanecarboxylic acid{5-[4-(1,1-dioxo-thiomorpholin-4-ylmethyl)-phenyl]-[1,2,4]triazolo[1,5-a]pyridin-2-yl}-amide(Compound 1), which has the chemical structure:

is disclosed in our earlier application WO2010/149769 (Menet C. J.,2010) as being an inhibitor of JAK and as being useful in the treatmentof inflammatory conditions, autoimmune diseases, proliferative diseases,allergy, transplant rejection, diseases involving impairment ofcartilage turnover, congenital cartilage malformations, and/or diseasesassociated with hypersecretion of IL6 or interferons. Hereafter thiscompound is named Compound 1. The data presented in WO 2010/149769demonstrate that despite similar in vitro activities, Compound 1 hasunexpectedly high in vivo potency compared with structurally similarcompounds.

An important characteristic of various bioactive substances (for examplebut without limitation pharmaceuticals, medicines and biocides, usuallyreferred to as drugs) is their “bio-availability” or activeconcentration in a form which can be absorbed and utilized by a targetorgan or organism. In many cases, the bioavailability is related to thedrug solubility in water.

To be of use as a therapeutic agent, the drug should be soluble in asuitable concentration range for the required period of time. Variousoptions are available to achieve these properties, including formulatingthe drug as a pill, capsules, solutions, or other similar formulations.Of particular interest are “zero-order release” drugs, in which the rateof drug release is constant. However, developing these systems can becomplicated and expensive.

Often, drugs in their free base form are poorly soluble in water, butthe presence of acidic sites (for example carboxylic acids, phenols,sulfonic acids) or basic sites (for example amino groups, basic nitrogencentres) can be used advantageously to produce salts of the drug. Theresulting ionic compounds become much more soluble in water by virtue oftheir ionic character and lower dissolution energy, and thus improvebioavailability. A guideline of 50 μg/mL for aqueous solubility isprovided by Lipinsky et al. (Lipinski, Lombardo, Dominy, & Feeney,2001).

Salt forming agents are available in large number, and salt selectionmust be carefully designed. The aim of the salt selection is to identifythe best salt form suitable for development, and is based primarily onfour main criteria: aqueous solubility at various pH, high degree ofcrystallinity, low hygroscopicity, and optimal chemical stability(Handbook of Pharmaceutical Salts: Properties, Selection and Use, Stahl,P. H. and Wermuth, C. G. Eds. Wiley-VCH, Weinheim, Germany, 2002).

If a suitable salt of a drug can be identified, further investigationsare required to identify whether there are alternative crystallineforms. The availability of such alternative forms is highlyunpredictable and can require a combination of intuition, carefulempirical design, perseverance and serendipity. On top of the challengesassociated with even finding one or more defined crystalline forms, theproperties of any crystalline forms thus discovered need to be carefullyevaluated to see if one or more of them is actually suitable forpharmaceutical development. Indeed, in a first aspect, crystallinity ofdrugs affects, among other physical and mechanical properties,solubility, dissolution rate, flowability, hardness, compressability,and melting point. In a second aspect, a crystalline form may haveadvantages over the amorphous form, for example, purification to thehigh degree of purity required by most regulatory authorities is moreefficient and therefore costs less for the crystalline form than for theamorphous solid. In addition, handling of the crystalline form isimproved over the amorphous form, which tends to be oily, or sticky, andin practice, drying of a crystalline material which has a well-defineddrying or desolvation temperature is more easily controlled, than forthe amorphous solid which has a greater affinity for organic solventsand variable drying temperature. Finally downstream processing of thecrystalline drug permits enhanced process control. In a third aspect,physical and chemical stability, and therefore shelf-life is alsoimproved for crystalline forms over amorphous forms.

Finally, pharmacokinetic and pharmacodynamic properties of a drug may belinked to a particular crystalline structural form, and it is paramountto produce and retain the same form from production to administration tothe patient. Therefore the obtention of salts, and/or crystalline formsover amorphous materials is highly desirable (Hilfiker, Blatter, & vonRaumer, 2006).

Thus the object of this invention is to disclose salt forms andpolymorphs of the salts of the invention, which have desirablepharmacological properties, and which are show improvements in theirpharmaceutical profile compared to the free base and/or amorphous formof the salt of the invention, in particular improved in vivo exposure.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the XRPD diffractogram of Compound 1 pattern 1.

FIG. 2 shows the XRPD diffractogram of Compound 1 pattern 3.

FIG. 3 shows the XRPD diffractogram of Compound 1 pattern 4.

FIG. 4 shows the XRPD diffractogram of Compound 1.HCl.

FIG. 5 shows the XRPD diffractogram of Compound 1.HCl.3H₂O.

FIG. 6 shows the Compound 1.HCl.3H₂O DVS analysis.

FIG. 7 shows the DSC trace of Compound 1.HCl.3H₂O.

FIG. 8 shows the XRPD diffractogram of Compound 1.HCl.MeOH.

FIG. 9 shows the XRPD diffractogram of Compound 1.HCl.1.5HCO₂H.

FIG. 10 shows the exposure of Compound 1 either as a free base or as anHCl.3H₂O salt upon daily po dosing.

FIG. 11 shows the Compound1.HCl.3H₂O crystal structure.

SUMMARY OF THE INVENTION

The present invention is based on the identification of novel salts andcrystalline forms of Compound 1, useful in the treatment and/orprophylaxis of inflammatory conditions, autoimmune diseases,proliferative diseases, allergy, transplant rejection, diseasesinvolving degradation and/or disruption of cartilage homeostasis,congenital cartilage malformations, and/or diseases associated withhypersecretion of IL6 or interferons. In particular, the salts of theinvention may act as inhibitors of JAK, and in more particularly ofJAK1. The present invention also provides methods for the production ofthese salts, pharmaceutical compositions comprising these salts andmethods for the treatment and/or prophylaxis of inflammatory conditions,autoimmune diseases, proliferative diseases, allergy, transplantrejection, diseases involving degradation and/or disruption of cartilagehomeostasis, congenital cartilage malformations, and/or diseasesassociated with hypersecretion of IL6 or interferons by administeringthe salts of the invention.

Accordingly, in one aspect the present invention provides a salt of acompound according to Formula (1) below (hereafter also named Compound1):

wherein said salt is formed with hydrobromic acid, hydrochloric acid,sulfuric acid, toluenesulfonic acid, benzenesulfonic acid, oxalic acid,maleic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonicacid, 1-2-ethane disulfonic acid, methanesulfonic acid, 2-hydroxyethanesulfonic acid, phosphoric acid, ethane sulfonic acid, malonicacid, 2-5-dihydroxybenzoic acid, or L-Tartaric acid.

Accordingly, in one aspect the present invention provides a salt of acompound according to Formula (1) below (hereafter also named Compound1):

wherein said salt is formed with hydrochloric acid

In another aspect of the invention, the salt of the invention is a[Compound 1.HCl.3H₂O] adduct in a solid crystalline form, wherein thecrystalline form is characterized at least by a powder X-ray diffractionpeak at any one or more of the following positions: 7.3, 8.4, 8.8, 10.7,12.0, 12.2, 13.2, 13.7, 14.5, 16.3, 16.7, 17.6, 19.3, 20.2, 20.6, 21.0,21.4, 21.8, 22.8, 23.4, 23.9, 24.5, 25.2, 25.7, 25.9, 26.4, 27.2, 27.7,28.3, 28.6, 28.9, 29.2, 29.6, 7 and 32.7° 2θ±0.2° 2θ.

In a particular aspect of the invention, the salt of the inventionexhibits improved solubility and exposure over the free base, which mayresult in an improved efficacy and a drug's lower dosage beingadministered. In turn, lowering the drug dosage level may potentiallylower toxicity that may occur via drug-drug interaction.

In a particular aspect, the salts of the invention are provided for usein the prophylaxis and/or treatment of inflammatory conditions,autoimmune diseases, proliferative diseases, allergy, transplantrejection, diseases involving degradation and/or disruption of cartilagehomeostasis, congenital cartilage malformations, and/or diseasesassociated with hypersecretion of IL6 or interferons.

In a further aspect, the present invention provides pharmaceuticalcompositions comprising a salt of the invention, and a pharmaceuticalcarrier, excipient or diluent. In a particular aspect, thepharmaceutical composition may additionally comprise furthertherapeutically active ingredients suitable for use in combination withthe salts of the invention. In a more particular aspect, the furthertherapeutically active ingredient is an agent for the treatment ofinflammatory conditions, autoimmune diseases, proliferative diseases,allergy, transplant rejection, diseases involving degradation and/ordisruption of cartilage homeostasis, congenital cartilage malformations,and/or diseases associated with hypersecretion of IL6 or interferons.

Moreover, the salts of the invention, useful in the pharmaceuticalcompositions and treatment methods disclosed herein, arepharmaceutically acceptable as prepared and used.

In a further aspect of the invention, this invention provides a methodof treating a mammal, in particular humans, afflicted with a conditionselected from among those listed herein, and particularly inflammatoryconditions, autoimmune diseases, proliferative diseases, allergy,transplant rejection, diseases involving degradation and/or disruptionof cartilage homeostasis, congenital cartilage malformations, and/ordiseases associated with hypersecretion of IL6 or interferons, whichmethod comprises administering an effective amount of the pharmaceuticalcomposition or salts of the invention as described herein.

The present invention also provides pharmaceutical compositionscomprising a salt of the invention, and a suitable pharmaceuticalcarrier, excipient or diluent for use in medicine. In a particularaspect, the pharmaceutical composition is for use in the prophylaxisand/or treatment of inflammatory conditions, autoimmune diseases,proliferative diseases, allergy, transplant rejection, diseasesinvolving degradation and/or disruption of cartilage homeostasis,congenital cartilage malformations, and/or diseases associated withhypersecretion of IL6 or interferons.

In additional aspects, this invention provides methods for synthesizingthe salts of the invention, with representative synthetic protocols andpathways disclosed later on herein.

Other objects and advantages will become apparent to those skilled inthe art from a consideration of the ensuing detailed description.

It will be appreciated that the salts of the invention may bemetabolized to yield biologically active metabolites.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The following terms are intended to have the meanings presentedtherewith below and are useful in understanding the description andintended scope of the present invention.

When describing the invention, which may include compounds,pharmaceutical compositions containing such compounds and methods ofusing such compounds and compositions, the following terms, if present,have the following meanings unless otherwise indicated. It should alsobe understood that when described herein any of the moieties definedforth below may be substituted with a variety of substituents, and thatthe respective definitions are intended to include such substitutedmoieties within their scope as set out below. Unless otherwise stated,the term ‘substituted’ is to be defined as set out below. It should befurther understood that the terms ‘groups’ and ‘radicals’ can beconsidered interchangeable when used herein.

The articles ‘a’ and ‘an’ may be used herein to refer to one or to morethan one (i.e. at least one) of the grammatical objects of the article.By way of example ‘an analogue’ means one analogue or more than oneanalogue.

‘Salt(s) of the invention’, and equivalent expressions, are meant toembrace salts of the compound according to Formula (I) (Compound 1) asherein described, which expression includes the pharmaceuticallyacceptable salts, and the solvates, e.g. hydrates, and the solvates ofthe pharmaceutically acceptable salts where the context so permits.

‘Pharmaceutically acceptable’ means approved or approvable by aregulatory agency of the Federal or a state government or thecorresponding agency in countries other than the United States, or thatis listed in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals, and more particularly, in humans.

‘Pharmaceutically acceptable vehicle’ refers to a diluent, adjuvant,excipient or carrier with which a salt of the invention is administered.

‘Solvate’ refers to forms of the compound that are associated with asolvent, usually by a solvolysis reaction. This physical associationincludes hydrogen bonding. Conventional solvents include water, ethanol,acetic acid and the like. The salts of the invention may be preparede.g. in crystalline form and may be solvated or hydrated. Suitablesolvates include pharmaceutically acceptable solvates, such as hydrates,and further include both stoichiometric solvates and non-stoichiometricsolvates. In certain instances the solvate will be capable of isolation,for example when one or more solvent molecules are incorporated in thecrystal lattice of the crystalline solid. ‘Solvate’ encompasses bothsolution-phase and isolable solvates. Representative solvates includehydrates, ethanolates and methanolates.

‘Subject’ includes humans. The terms ‘human’, ‘patient’ and ‘subject’are used interchangeably herein.

‘Effective amount’ means the amount of a salt of the invention that,when administered to a subject for treating a disease, is sufficient toeffect such treatment for the disease. The “effective amount” can varydepending on the compound, the disease and its severity, and the age,weight, etc., of the subject to be treated.

‘Preventing’ or ‘prevention’ refers to a reduction in risk of acquiringor developing a disease or disorder (i.e. causing at least one of theclinical symptoms of the disease not to develop in a subject that may beexposed to a disease-causing agent, or predisposed to the disease inadvance of disease onset.

The term ‘prophylaxis’ is related to ‘prevention’, and refers to ameasure or procedure the purpose of which is to prevent, rather than totreat or cure a disease. Non-limiting examples of prophylactic measuresmay include the administration of vaccines; the administration of lowmolecular weight heparin to hospital patients at risk for thrombosisdue, for example, to immobilization; and the administration of ananti-malarial agent such as chloroquine, in advance of a visit to ageographical region where malaria is endemic or the risk of contractingmalaria is high.

‘Treating’ or ‘treatment’ of any disease or disorder refers, in oneembodiment, to ameliorating the disease or disorder (i.e. arresting thedisease or reducing the manifestation, extent or severity of at leastone of the clinical symptoms thereof). In another embodiment ‘treating’or ‘treatment’ refers to ameliorating at least one physical parameter,which may not be discernible by the subject. In yet another embodiment,‘treating’ or ‘treatment’ refers to modulating the disease or disorder,either physically, (e.g. stabilization of a discernible symptom),physiologically, (e.g. stabilization of a physical parameter), or both.In a further embodiment, “treating” or “treatment” relates to slowingthe progression of the disease.

As used herein the term ‘inflammatory diseases’ refers to the group ofconditions including, rheumatoid arthritis, osteoarthritis, juvenileidiopathic arthritis, psoriasis, psoriatic arthritis, allergic airwaydisease (e.g. asthma, rhinitis), chronic obstructive pulmonary disease(COPD), inflammatory bowel diseases (e.g. Crohn's disease, Whipple,chronic ulcerative colitis, or colitis), endotoxin-driven disease states(e.g. complications after bypass surgery or chronic endotoxin statescontributing to e.g. chronic cardiac failure), and related diseasesinvolving cartilage, such as that of the joints. Particularly the termrefers to rheumatoid arthritis, osteoarthritis, allergic airway disease(e.g. asthma), chronic obstructive pulmonary disease (COPD) andinflammatory bowel diseases. More particularly the term refers torheumatoid arthritis, and inflammatory bowel diseases (e.g. Crohn'sdisease, Whipple, chronic ulcerative colitis, or colitis).

As used herein the term ‘autoimmune disease(s)’ refers to the group ofdiseases including obstructive airways disease, including conditionssuch as COPD, asthma (e.g. intrinsic asthma, extrinsic asthma, dustasthma, infantile asthma) particularly chronic or inveterate asthma (forexample late asthma and airway hyperresponsiveness), bronchitis,including bronchial asthma, systemic lupus erythematosus (SLE),cutaneous lupus erythematosus, lupus nephritis, dermatomyositis,Sjogren's syndrome, multiple sclerosis, psoriasis, dry eye disease, typeI diabetes mellitus and complications associated therewith, atopiceczema (atopic dermatitis), thyroiditis (Hashimoto's and autoimmunethyroiditis), contact dermatitis and further eczematous dermatitis,inflammatory bowel disease (e.g. Crohn's disease, Whipple, chroniculcerative colitis, or colitis), atherosclerosis and amyotrophic lateralsclerosis. Particularly the term refers to COPD, asthma, systemic lupuserythematosus, type I diabetes mellitus and inflammatory bowel disease.

As used herein the term ‘proliferative disease(s)’ refers to conditionssuch as cancer (e.g. uterine leiomyosarcoma or prostate cancer),myeloproliferative disorders (e.g. polycythemia vera, essentialthrombocytosis and myelofibrosis), leukemia (e.g. acute myeloidleukaemia, acute and chronic lymphoblastic leukemia), multiple myeloma,psoriasis, restenosis, scleroderma or fibrosis. In particular the termrefers to cancer, leukemia, multiple myeloma and psoriasis.

As used herein, the term ‘cancer’ refers to a malignant or benign growthof cells in skin or in body organs, for example but without limitation,breast, prostate, lung, kidney, pancreas, stomach or bowel. A cancertends to infiltrate into adjacent tissue and spread (metastasise) todistant organs, for example to bone, liver, lung or the brain. As usedherein the term cancer includes both metastatic tumour cell types (suchas but not limited to, melanoma, lymphoma, leukaemia, fibrosarcoma,rhabdomyosarcoma, and mastocytoma) and types of tissue carcinoma (suchas but not limited to, colorectal cancer, prostate cancer, small celllung cancer and non-small cell lung cancer, breast cancer, pancreaticcancer, bladder cancer, renal cancer, gastric cancer, glioblastoma,primary liver cancer, ovarian cancer, prostate cancer and uterineleiomyosarcoma). In particular, the term “cancer” refers to acutelymphoblastic leukemia, acute myeloidleukemia, adrenocortical carcinoma,anal cancer, appendix cancer, astrocytomas, atypical teratoid/rhabdoidtumor, basal cell carcinoma, bile duct cancer, bladder cancer, bonecancer (osteosarcoma and malignant fibrous histiocytoma), brain stemglioma, brain tumors, brain and spinal cord tumors, breast cancer,bronchial tumors, Burkitt lymphoma, cervical cancer, chronic lymphocyticleukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer,craniopharyngioma, cutaneous T-Cell lymphoma, embryonal tumors,endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer,ewing sarcoma family of tumors, eye cancer, retinoblastoma, gallbladdercancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor,gastrointestinal stromal tumor (GIST), gastrointestinal stromal celltumor, germ cell tumor, glioma, hairy cell leukemia, head and neckcancer, hepatocellular (liver) cancer, hypopharyngeal cancer,intraocular melanoma, islet cell tumors (endocrine pancreas), Kaposisarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer,leukemia, Acute lymphoblastic leukemia, acute myeloid leukemia, chroniclymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia,liver cancer, non-small cell lung cancer, small cell lung cancer,Burkitt lymphoma, cutaneous T-celllymphoma, Hodgkin lymphoma,non-Hodgkin lymphoma, lymphoma, Waldenstrom macroglobulinemia,medulloblastoma, medulloepithelioma, melanoma, mesothelioma, mouthcancer, chronic myelogenous leukemia, myeloid leukemia, multiplemyeloma, asopharyngeal cancer, neuroblastoma, non-small cell lungcancer, oral cancer, oropharyngeal cancer, osteosarcoma, malignantfibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer,ovarian germ cell tumor, ovarian low malignant potential tumor,pancreatic cancer, papillomatosis, parathyroid cancer, penile cancer,pharyngeal cancer, pineal parenchymal tumors of intermediatedifferentiation, pineoblastoma and supratentorial primitiveneuroectodermal tumors, pituitary tumor, plasma cell neoplasm/multiplemyeloma, pleuropulmonary blastoma, primary central nervous systemlymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer,retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Ewingsarcoma family of tumors, sarcoma, Sezary syndrome, skin cancer, smallcell Lung cancer, small intestine cancer, soft tissue sarcoma, squamouscell carcinoma, stomach (gastric) cancer, supratentorial primitiveneuroectodermal tumors, T-cell lymphoma, testicular cancer, throatcancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer,uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer,Waldenstrom macroglobulinemia, and Wilms tumor. More particularly, thecancer is selected from breast cancer, endometrial and cervical cancer,lung cancer, ovarian cancer, prostate cancer, hepatic cancer, andpancreatic cancer.

As used herein the term ‘leukemia’ refers to neoplastic diseases of theblood and blood forming organs. Such diseases can cause bone marrow andimmune system dysfunction, which renders the host highly susceptible toinfection and bleeding. In particular the term leukemia refers to acutemyeloid leukaemia (AML), and acute lymphoblastic leukemia (ALL) andchronic lymphoblastic leukaemia (CLL).

As used herein the term ‘allergy’ refers to the group of conditionscharacterized by a hypersensitivity disorder of the immune systemincluding, allergic airway disease (e.g. asthma, rhinitis), sinusitis,eczema and hives, as well as food allergies or allergies to insectvenom.

As used herein the term ‘asthma’ as used herein refers to any disorderof the lungs characterized by variations in pulmonary gas flowassociated with airway constriction of whatever cause (intrinsic,extrinsic, or both; allergic or non-allergic). The term asthma may beused with one or more adjectives to indicate the cause.

As used herein the term ‘transplant rejection’ refers to the acute orchronic rejection of cells, tissue or solid organ allo- or xenografts ofe.g. pancreatic islets, stem cells, bone marrow, skin, muscle, cornealtissue, neuronal tissue, heart, lung, combined heart-lung, kidney,liver, bowel, pancreas, trachea or oesophagus, or graft-versus-hostdiseases.

As used herein the term ‘diseases involving degradation and/ordisruption of cartilage homeostasis’ includes conditions such asosteoarthritis, psoriatic arthritis, juvenile rheumatoid arthritis,gouty arthritis, septic or infectious arthritis, reactive arthritis,reflex sympathetic dystrophy, algodystrophy, achondroplasia, Paget'sdisease, Tietze syndrome or costal chondritis, fibromyalgia,osteochondritis, neurogenic or neuropathic arthritis, arthropathy,sarcoidosis, amylosis, hydarthrosis, periodical disease, rheumatoidspondylitis, endemic forms of arthritis like osteoarthritis deformansendemica, Mseleni disease and Handigodu disease; degeneration resultingfrom fibromyalgia, systemic lupus erythematosus, scleroderma andankylosing spondylitis.

As used herein the term ‘congenital cartilage malformation(s)’ includesconditions such as hereditary chondrolysis, chondrodysplasias andpseudochondrodysplasias, in particular, but without limitation,microtia, anotia, metaphyseal chondrodysplasia, and related disorders.

As used herein the term ‘disease(s) associated with hypersecretion ofIL6’ includes conditions such as Castleman's disease, multiple myeloma,psoriasis, Kaposi's sarcoma and/or mesangial proliferativeglomerulonephritis.

As used herein the term ‘disease(s) associated with hypersecretion ofinterferons’ includes conditions such as systemic and cutaneous lupuserythematosis, lupus nephritis, dermatomyositis, Sjogren's syndrome,psoriasis, rheumatoid arthritis.

When ranges are referred to herein, for example but without limitation,C₁₋₈ alkyl, the citation of a range should be considered arepresentation of each member of said range.

As used herein, the term ‘isotopic variant’ refers to a compound thatcontains unnatural proportions of isotopes at one or more of the atomsthat constitute such compound. For example, an ‘isotopic variant’ of acompound can contain one or more non-radioactive isotopes, such as forexample, deuterium (²H or D), carbon-13 (¹³C), nitrogen-15 (¹⁵N), or thelike. It will be understood that, in a compound where such isotopicsubstitution is made, the following atoms, where present, may vary, sothat for example, any hydrogen may be ²H/D, any carbon may be ¹³C, orany nitrogen may be ¹⁵N, and that the presence and placement of suchatoms may be determined within the skill of the art. Likewise, theinvention may include the preparation of isotopic variants withradioisotopes, in the instance for example, where the resultingcompounds may be used for drug and/or substrate tissue distributionstudies. The radioactive isotopes tritium, i.e. ³H, and carbon-14, i.e.¹⁴C, are particularly useful for this purpose in view of their ease ofincorporation and ready means of detection. Further, compounds may beprepared that are substituted with positron emitting isotopes, such as¹¹C, ¹⁸F, ¹⁵O and ¹³N and would be useful in Positron EmissionTopography (PET) studies for examining substrate receptor occupancy.

All isotopic variants of the compounds provided herein, radioactive ornot, are intended to be encompassed within the scope of the invention.

‘Tautomers’ refer to compounds that are interchangeable forms of aparticular compound structure, and that vary in the displacement ofhydrogen atoms and electrons. Thus, two structures may be in equilibriumthrough the movement of π electrons and an atom (usually H). Forexample, enols and ketones are tautomers because they are rapidlyinterconverted by treatment with either acid or base. Another example oftautomerism is the aci- and nitro-forms of phenylnitromethane, that arelikewise formed by treatment with acid or base.

Tautomeric forms may be relevant to the attainment of the optimalchemical reactivity and biological activity of a compound of interest.

It will be appreciated that salts of the invention may be metabolized toyield biologically active metabolites.

THE INVENTION

The present invention relates to salts of the compoundcyclopropanecarboxylic acid{5-[4-(1,1-dioxo-thiomorpholin-4-ylmethyl)-phenyl]-[1,2,4]triazolopyridin-2-yl}-amide (Compound 1), and methods for the preparation ofsuch salts, which are useful in the prophylaxis and/or treatment ofinflammatory conditions, autoimmune diseases, proliferative diseases,allergy, transplant rejection, diseases involving degradation and/ordisruption of cartilage homeostasis, congenital cartilage malformations,and/or diseases associated with hypersecretion of IL6 or interferons. Inparticular, the salts of the invention inhibit JAK, a family of tyrosinekinases, and more particularly JAK1.

The present invention also provides methods for the prophylaxis and/ortreatment of diseases including inflammatory conditions, autoimmunediseases, proliferative diseases, allergy, transplant rejection,diseases involving degradation and/or disruption of cartilagehomeostasis, congenital cartilage malformations, and/or diseasesassociated with hypersecretion of IL6 or interferons by administering asalt of the invention, or a pharmaceutical composition containing saidsalt.

Accordingly, in one aspect the present invention provides a salt of acompound according to Formula (I) (Compound 1) below:

wherein said salt is formed with a salt forming agent selected fromhydrobromic acid, hydrochloric acid, sulfuric acid, toluenesulfonicacid, benzenesulfonic acid, oxalic acid, maleic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-2-ethanedisulfonic acid, methanesulfonic acid, 2-hydroxy ethanesulfonic acid,phosphoric acid, ethane sulfonic acid, malonic acid,2-5-dihydroxybenzoic acid, and L-Tartaric acid.

In one embodiment, the salt of the invention is one formed with a saltforming agent selected from hydrobromic acid, and hydrochloric acid, inparticular hydrochloric acid.

In one embodiment, the salt of the invention is one formed with a saltforming agent selected from oxalic acid, maleic acid, or malonic acid,in particular maleic acid.

In one embodiment, the salt of the invention is one formed with a saltforming agent selected from toluenesulfonic acid, benzenesulfonic acid,naphthalene-2-sulfonic acid, and ethanesulfonic acid; in particulartoluenesulfonic acid, and benzenesulfonic acid, more particularlytoluenesulfonic acid, and most particularly para-toluenesulfonic acid.

In one embodiment, the salt of the invention is a 3:1 to 1:3 [Compound1:salt forming agent] adduct. In a particular embodiment, the salt ofthe invention is a 1:1 [Compound 1:salt forming agent] adduct. In a moreparticular embodiment, the salt forming agent is selected fromhydrobromic acid, hydrochloric acid, toluenesulfonic acid, and maleicacid. In a most particular embodiment, the salt forming agent ishydrochloric acid.

In one embodiment, the salt of the invention is a solvate. In aparticular embodiment, the salt of the invention is a mono-, di-, ortrisolvate. In a most particular embodiment, the salt of the inventionis a trisolvate. Alternatively, the salt of the invention is not asolvate.

In one embodiment, the salt of the invention is a hydrate. In a moreparticular embodiment, the salt of the invention is a mono-, di-, ortrihydrate. In a most particular embodiment, the salt of the inventionis a trihydrate. Alternatively, the salt of the invention is anhydrous.

In one embodiment, the salt of the invention is a [Compound 1:Saltforming agent:Solvent] adduct. In a particular embodiment, the salt ofthe invention is a 1:1:0 to 1:1:4 [Compound 1:Salt formingagent:Solvent] adduct. In a more particular embodiment, the salt of theinvention is a 1:1:0, 1:1:1, 1:1:1.5, 1:1:2, or 1:1:3 [Compound 1:Saltforming agent:Solvent] adduct. In a most particular embodiment, the saltof the invention is a 1:1:3 [Compound 1:Salt forming agent:Solvent]adduct.

In another embodiment, the salt of the invention is a [Compound1:HCl:Solvent] adduct. In a particular embodiment, the salt of theinvention is a 1:1:0 to 1:1:4 [Compound 1:HCl:Solvent] adduct. In a moreparticular embodiment, the salt of the invention is a 1:1:0, 1:1:1,1:1:1.5, 1:1:2, or 1:1:3 [Compound 1:HCl:Solvent] adduct. In a mostparticular embodiment, the salt of the invention is a 1:1:3 [Compound1:HCl:Solvent] adduct. In a further most particular embodiment, thesolvent is selected from H₂O, MeOH, and HCO₂H.

In another embodiment, the salt of the invention is a [Compound1:HCl:H₂O] adduct. In a particular embodiment, the salt of the inventionis a 1:1:0 to 1:1:4 [Compound 1:HCl:H₂O] adduct. In a more particularembodiment, the salt of the invention is a 1:1:0, 1:1:1, 1:1:1.5, 1:1:2,or 1:1:3 [Compound 1:HCl:H₂O] adduct. In a most particular embodiment,the salt of the invention is a 1:1:3 [Compound 1:HCl:H₂O] adduct.

In one embodiment, the salt of the invention exhibits peaks on a XRPDspectrum.

In one embodiment, the salt of the invention is in a crystalline form.

In one embodiment, the salt of the invention is a 1:1:0 [Compound1:HCl:H₂O] adduct in a solid crystalline form, wherein the crystallineform is characterized at least by a powder X-ray diffraction peak at anyone or more of the following positions: 7.4, 8.9, 12.4, 14.8, 15.1,16.9, 17.6, 19.4, 20.7, 21.1, 22.8, 24.9, 26.0, 28.6, 29.8, and 32.6°2θ±0.2° 2θ.

In one embodiment, the salt of the invention is a 1:1:0 [Compound1:HCl:H₂O] adduct in a solid crystalline form, wherein the crystallineform is characterized at least by a powder X-ray diffraction peak atleast at 5, 10, 15, or more of the following positions: 7.4, 8.9, 12.4,14.8, 15.1, 16.9, 17.6, 19.4, 20.7, 21.1, 22.8, 24.9, 26.0, 28.6, 29.8,and 32.6° 20±0.2° 20.

In one embodiment, the salt of the invention is a 1:1:0 [Compound1:HCl:H₂O] adduct in a solid crystalline form, wherein the crystallineform is characterized by a powder X-ray diffraction peak in all of thefollowing positions: 7.4, 8.9, 12.4, 14.8, 15.1, 16.9, 17.6, 19.4, 20.7,21.1, 22.8, 24.9, 26.0, 28.6, 29.8, and 32.6° 2θ±0.2° 2θ. In aparticular embodiment, the salt of the invention is characterized by theXRPD pattern expressed in terms of 2 theta angles as shown on Error!Reference source not found.

In one embodiment, the salt of the invention is a 1:1:3 [Compound1:HCl:H₂O] adduct in a solid crystalline form, wherein the crystallineform is characterized at least by a powder X-ray diffraction peak at anyone or more of the following positions: 7.3, 8.4, 8.8, 10.7, 12.0, 12.2,13.2, 13.7, 14.5, 16.3, 16.7, 17.6, 19.3, 20.2, 20.6, 21.0, 21.4, 21.8,22.8, 23.4, 23.9, 24.5, 25.2, 25.7, 25.9, 26.4, 27.2, 27.7, 28.3, 28.6,28.9, 29.2, 29.6, and 32.7° 2θ±0.2° 2θ.

In a particular embodiment, the salt of the invention is a 1:1:3[Compound 1:HCl:H₂O] adduct in a solid crystalline form, wherein thecrystalline form is characterized at least by a powder X-ray diffractionpeak at least at 5, 10, 15, 20, 25, 30 or more of the followingpositions: 7.3, 8.4, 8.8, 10.7, 12.0, 12.2, 13.2, 13.7, 14.5, 16.3,16.7, 17.6, 19.3, 20.2, 20.6, 21.0, 21.4, 21.8, 22.8, 23.4, 23.9, 24.5,25.2, 25.7, 25.9, 26.4, 27.2, 27.7, 28.3, 28.6, 28.9, 29.2, 29.6, and32.7° 2θ±0.2° 2θ.

In one embodiment, the salt of the invention is a 1:1:3 [Compound1:HCl:H₂O] adduct in a solid crystalline form, wherein the crystallineform is characterized by a powder X-ray diffraction peak in all of thefollowing positions: 7.3, 8.4, 8.8, 10.7, 12.0, 12.2, 13.2, 13.7, 14.5,16.3, 16.7, 17.6, 19.3, 20.2, 20.6, 21.0, 21.4, 21.8, 22.8, 23.4, 23.9,24.5, 25.2, 25.7, 25.9, 26.4, 27.2, 27.7, 28.3, 28.6, 28.9, 29.2, 29.6,and 32.7° 2θ±0.2° 2θ. In a particular embodiment, the salt of theinvention is characterized by the XRPD pattern expressed in terms of 2theta angles as shown on Error! Reference source not found.

In one embodiment, the 1:1:3 [Compound 1:HCl:H₂O] adduct in a solidcrystalline form has a particle size of less than 1000 μM, as measuredby laser diffraction (Table II). In a particular embodiment, the 1:1:3[Compound 1:HCl:H₂O] adduct in a solid crystalline form has a particlesize between 50 μm and 800 μm. In a more particular embodiment, the1:1:3 [Compound 1:HCl:H₂O] adduct in a solid crystalline form has aparticle size between 200 μm and 600 μm.

In one embodiment, the salt of the invention is a 1:1:1 [Compound1:HCl:MeOH] adduct in a solid crystalline form, wherein the crystallineform is characterized at least by a powder X-ray diffraction peak at anyone or more of the following positions: 7.1, 14.4, 16.6, 17.3, 18.9,23.4, 24.8, and 29.0° 2θ±0.2° 2θ.

In a particular embodiment, the salt of the invention is a 1:1:1[Compound 1:HCl:MeOH] adduct in a solid crystalline form, wherein thecrystalline form is characterized at least by a powder X-ray diffractionpeak at least at 3, 5, 7 or more of the following positions: 7.1, 14.4,16.6, 17.3, 18.9, 23.4, 24.8, and 29.0° 2θ±0.2° 2θ.

In one embodiment, the salt of the invention is a 1:1:1 [Compound1:HCl:MeOH] adduct in a solid crystalline form, wherein the crystallineform is characterized by a powder X-ray diffraction peak in all of thefollowing positions: 7.1, 14.4, 16.6, 17.3, 18.9, 23.4, 24.8, and 29.0°2θ±0.2° 2θ. In a particular embodiment, the salt of the invention ischaracterized by the XRPD pattern expressed in terms of 2 theta anglesas shown on Error! Reference source not found.

In one embodiment, the salt of the invention is a 1:1:1.5 [Compound1:HCl:HCO₂H] adduct in a solid crystalline form, wherein the crystallineform is characterized at least by a powder X-ray diffraction peak at anyone or more of the following positions: 7.1, 14.4, 14.8, 16.4, 17.4,18.6, 20.8, 23.4, 24.5, 24.9, and 29.0° 2θ±0.2° 2θ.

In one embodiment, the salt of the invention is a 1:1:1.5 [Compound1:HCl:HCO₂H] adduct in a solid crystalline form, wherein the crystallineform is characterized at least by a powder X-ray diffraction peak atleast at 3, 5, 7, 9 or more of the following positions: 7.1, 14.4, 14.8,16.4, 17.4, 18.6, 20.8, 23.4, 24.5, 24.9, and 29.0° 2θ±0.2° 2θ.

In one embodiment, the salt of the invention is a 1:1:1.5 [Compound1:HCl:HCO₂H] adduct in a solid crystalline form, wherein the crystallineform is characterized by a powder X-ray diffraction peak in all of thefollowing positions: 7.1, 14.4, 14.8, 16.4, 17.4, 18.6, 20.8, 23.4,24.5, 24.9, and 29.0° 2θ±0.2° 2θ. In a particular embodiment, the saltof the invention is characterized by the XRPD pattern expressed in termsof 2 theta angles as shown on Error! Reference source not found.

In one embodiment, a salt of the invention is obtained by combining aCompound 1, with an acid selected from hydrobromic acid, hydrochloricacid, sulfuric acid, toluenesulfonic acid, benzenesulfonic acid, oxalicacid, maleic acid, naphthalene-2-sulfonic acid,naphthalene-1,5-disulfonic acid, 1-2-ethane disulfonic acid,methanesulfonic acid, 2-hydroxy ethanesulfonic acid, phosphoric acid,ethane sulfonic acid, malonic acid, 2-5-dihydroxybenzoic acid, andL-Tartaric acid, in an inert solvent and precipitating said salt fromsaid solvent. In a particular embodiment, the salt of the invention isobtained by adding Compound 1 and a salt forming agent in a suitablesolvent in order to achieve full dissolution, followed by a controlledsolvent evaporation in order to achieve supersaturation, and thuscrystallization of the corresponding salt.

In one embodiment, the salt of the invention is obtained by mixingCompound 1, and an acid in a molar ratio of between 5:1 and 1:5 ofCompound 1:acid. In a particular embodiment, the salt of the inventionis obtained by mixing Compound 1, and an acid in a molar ratio ofbetween 2:1 and 1:2 of Compound 1:acid. In a more particular embodiment,the salt of the invention is obtained by mixing a Compound 1 and an acidin a molar ratio of 1:1.

In another particular embodiment, the solvent for the preparation of thesalt of the invention is selected from ketones, alcohols, esters, C₁₋₁₀alkyl, C₃₋₇ cycloalkyl, C₆₋₁₀ monocyclic or fused bicyclic aryl,sulfoxide, C₁₋₁₀ alkylnitrile, C₁₋₁₀ linear, branched or cyclic ethers,and C₁₋₁₀ haloalkyl, In a particular embodiment, the solvent for thepreparation of the salt of the invention is selected from acetone,anisole, butanol, butyl acetate, TBME, DMSO, ethanol, ethyl acetate,heptane, isopropyl acetate, MEK, isopropyl acetate, MeCN, cyclohexane,DCM, dioxane, methanol, nitromethane, THF, methyl THF, toluene, water,10% aqueous acetone, 10% aqueous THF, and 10% methanol. In a particularembodiment, the solvent for the preparation of the salt of the inventionis selected from dioxane, THF, acetone, DCM, and MeOH.

In another aspect is provided a method for preparing the salt of theinvention comprising the steps of

-   -   i) reacting Compound 1, with an acid selected from hydrobromic        acid, hydrochloric acid, sulfuric acid, toluenesulfonic acid,        benzenesulfonic acid, oxalic acid, maleic acid,        naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid,        1-2-ethane disulfonic acid, methanesulfonic acid, 2-hydroxy        ethanesulfonic acid, phosphoric acid, ethane sulfonic acid,        malonic acid, 2-5-dihydroxybenzoic acid, and L-Tartaric acid, in        an inert solvent; and    -   ii) precipitating the said salt from the said solvent.

In a further aspect, is provided a salt of the invention obtainable byor obtained by the aforementioned method.

In one embodiment, with respect to the preparation of the salt of theinvention, the inert solvent is selected from dioxane, THF, acetone,DCM, and MeOH.

In one embodiment, with respect to the preparation of the salt of theinvention, the inert solvent is DCM.

In one embodiment, with respect to the preparation of the salt of theinvention, the inert solvent is selected from iPrOH/water, iPrOH, iBuOH,and tBuOH.

In one embodiment is provided a method for preparing a [Compound1:HCl:3H₂O] adduct in a solid crystalline form comprising the steps of:

-   -   i) Stirring the Compound 1 with water,    -   ii) Adding aqueous HCl,    -   iii) Stirring further the mixture obtained at step ii),    -   iv) Cooling the mixture of step iii) to 15° C.,    -   v) Continuing stirring for at most 24 h at 15° C.,    -   vi) separating the resulting solid by filtration obtained in the        previous step v), and    -   vii) drying under nitrogen for at least 4 h said resulting solid        obtained in the previous step vi).

In a particular embodiment is provided a method for preparing a[Compound 1:HCl:3H₂O] adduct in a solid crystalline form comprising thesteps of:

-   -   i) Stirring the Compound 1 with water at 50° C.,    -   ii) Adding aqueous HCl,    -   iii) Stirring the mixture obtained at step ii) further at 50° C.        for 15 min,    -   iv) Cooling the mixture of step iii) to 15° C.,    -   v) Continuing stirring for 12 h to 24 h at 15° C.,    -   vi) separating the resulting solid by filtration obtained in the        previous step v), and    -   vii) drying under nitrogen for at least 4 h said resulting solid        obtained in the previous step vi).

In another embodiment is provided a method for preparing a [Compound1:HCl:3H₂O] adduct in a solid crystalline form comprising the steps of:

-   -   i) mixing Compound 1 suspended in DCM, with MeOH,    -   ii) adding water under stirring,    -   iii) separating the organic layer,    -   iv) adding a solution of HCl to the organic layer obtained in        the previous step iii),    -   v) separating the resulting solid by filtration obtained in the        previous step iv),    -   vi) drying said resulting solid obtained in the previous step        v),    -   vii) adding the solid obtained in the previous step vi) to a        formic acid/water solution, under stirring,    -   viii) adding water to the solution obtained in the previous step        vii),    -   ix) separating by filtration the resulting solid obtained in the        previous step viii), and    -   x) drying the resulting solid obtained in the previous step ix).

In another embodiment is provided a method for preparing a [Compound1:HCl:3H₂O] adduct in a solid crystalline form comprising the steps of:

-   -   i) mixing Compound 1 suspended in DCM, with MeOH at 35° C.,    -   ii) adding water under stirring at 35° C. for at least 15 min,    -   iii) separating the organic layer,    -   iv) adding a 10% w/w solution of HCl in MeOH to the organic        layer obtained in the previous step iii),    -   v) separating the resulting solid by filtration obtained in the        previous step iv),    -   vi) drying said resulting solid obtained in the previous step        v),    -   vii) adding the solid obtained in the previous step vi) to a        1.6/0.4 formic acid/water solution, under stirring at 55° C.    -   viii) adding water to the solution obtained in the previous step        vii),    -   ix) separating by filtration the resulting solid obtained in the        previous step viii), and    -   x) drying the resulting solid obtained in the previous step ix).

In another embodiment is provided a method for preparing a [Compound1:HCl:3H₂O] adduct in a solid crystalline form comprising the steps of:

-   -   i) reacting Compound 1 suspended in DCM, with MeOH and        trimercaptotriazine trisodium,    -   ii) filtering the resulting suspension,    -   iii) adding water under stirring,    -   iv) separating the organic layer,    -   v) adding a solution of HCl to the organic layer obtained at        step iv),    -   vi) separating the resulting solid by filtration obtained at        step v),    -   vii) drying said resulting solid obtained at step vi),    -   viii) adding the solid obtained at step vii) to a formic        acid/water solution, under stirring,    -   ix) adding water to the solution of step viii),    -   x) separating by filtration the resulting solid obtained at step        ix), and    -   xi) drying the resulting solid obtained at step x).

In another aspect is provided a method for preparing a [Compound1:HCl:3H₂O] adduct in a solid crystalline form comprising the steps of:

-   -   i) reacting Compound 1 suspended in DCM, with MeOH and        trimercaptotriazine trisodium at 35° C. for at least 5 h,    -   ii) filtering the resulting suspension,    -   iii) adding water under stirring at 35° C. for at least 15 min,    -   iv) separating the organic layer,    -   v) adding a 10% w/w solution of HCl in MeOH to the organic layer        obtained at step iv),    -   vi) separating the resulting solid by filtration obtained at        step v),    -   vii) drying said resulting solid obtained at step vi),    -   viii) adding the solid obtained at step vii) to a 1.6/0.4 formic        acid/water solution, under stirring at 55° C.,    -   ix) adding water to the solution of step viii),    -   x) separating by filtration the resulting solid obtained at step        ix), and    -   xi) drying the resulting solid obtained at step x).

Alternatively, the exclusion of one or more of the specified variablesfrom a group or an embodiment, or combinations thereof is alsocontemplated by the present invention.

Pharmaceutical Compositions

When employed as a pharmaceutical, a salt of the invention is typicallyadministered in the form of a pharmaceutical composition. Suchcompositions can be prepared in a manner well known in thepharmaceutical art and comprise at least one active salt of theinvention.

Generally, a salt of the invention is administered in a pharmaceuticallyeffective amount. The amount of salt of the invention actuallyadministered will typically be determined by a physician, in the lightof the relevant circumstances, including the condition to be treated,the chosen route of administration, the actual salt of the inventionadministered, the age, weight, and response of the individual patient,the severity of the patient's symptoms, and the like.

The pharmaceutical compositions of this invention can be administered bya variety of routes including oral, rectal, transdermal, subcutaneous,intra-articular, intravenous, intramuscular, and intranasal. Dependingon the intended route of delivery, a salt of the invention is preferablyformulated as either injectable or oral compositions or as salves, aslotions or as patches all for transdermal administration.

The compositions for oral administration can take the form of bulkliquid solutions or suspensions, or bulk powders. More commonly,however, the compositions are presented in unit dosage forms tofacilitate accurate dosing. The term ‘unit dosage forms’ refers tophysically discrete units suitable as unitary dosages for human subjectsand other mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient, vehicle orcarrier. Typical unit dosage forms include prefilled, premeasuredampules or syringes of the liquid compositions or pills, tablets,capsules or the like in the case of solid compositions. In suchcompositions, the salt of the invention is usually a minor component(from about 0.1 to about 50% by weight or preferably from about 1 toabout 40% by weight) with the remainder being various vehicles orcarriers and processing aids helpful for forming the desired dosingform.

Liquid forms suitable for oral administration may include a suitableaqueous or non-aqueous vehicle with buffers, suspending and dispensingagents, colorants, flavours and the like. Solid forms may include, forexample, any of the following ingredients, or salt of the inventions ofa similar nature: a binder such as microcrystalline cellulose, gumtragacanth or gelatine; an excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel, or corn starch; alubricant such as magnesium stearate; a glidant such as colloidalsilicon dioxide; a sweetening agent such as sucrose or saccharin; or aflavouring agent such as peppermint or orange flavouring.

Injectable compositions are typically based upon injectable sterilesaline or phosphate-buffered saline or other injectable carriers knownin the art. As before, the active salt of the invention according toFormula I in such compositions is typically a minor component, oftenbeing from about 0.05 to 10% by weight with the remainder being theinjectable carrier and the like.

Transdermal compositions are typically formulated as a topical ointmentor cream containing the active ingredient(s), generally in an amountranging from about 0.01 to about 20% by weight, preferably from about0.1 to about 20% by weight, preferably from about 0.1 to about 10% byweight, and more preferably from about 0.5 to about 15% by weight. Whenformulated as an ointment, the active ingredients will typically becombined with either a paraffinic or a water-miscible ointment base.Alternatively, the active ingredients may be formulated in a cream with,for example an oil-in-water cream base. Such transdermal formulationsare well-known in the art and generally include additional ingredientsto enhance the dermal penetration of stability of the active ingredientsor the formulation. All such known transdermal formulations andingredients are included within the scope of this invention.

A salt of the invention can also be administered by a transdermaldevice. Accordingly, transdermal administration can be accomplishedusing a patch either of the reservoir or porous membrane type, or of asolid matrix variety.

The above-described components for orally administrable, injectable ortopically administrable compositions are merely representative. Othermaterials as well as processing techniques and the like are set forth inPart 8 of Remington's Pharmaceutical Sciences, 17^(th) edition, 1985,Mack Publishing Company, Easton, Pa., which is incorporated herein byreference.

A salt of the invention can also be administered in sustained releaseforms or from sustained release drug delivery systems. A description ofrepresentative sustained release materials can be found in Remington'sPharmaceutical Sciences.

The following formulation examples illustrate representativepharmaceutical compositions that may be prepared in accordance with thisinvention. The present invention, however, is not limited to thefollowing pharmaceutical compositions.

Formulation 1—Tablets

A salt of the invention may be admixed as a dry powder with a drygelatin binder in an approximate 1:2 weight ratio. A minor amount ofmagnesium stearate may be added as a lubricant. The mixture may beformed into 240-270 mg tablets (80-90 mg of active salt of the inventionper tablet) in a tablet press.

Formulation 2—Capsules

A salt of the invention may be admixed as a dry powder with a starchdiluent in an approximate 1:1 weight ratio. The mixture may be filledinto 250 mg capsules (125 mg of active salt of the invention percapsule).

Formulation 3—Liquid

A salt of the invention (125 mg), may be admixed with sucrose (1.75 g)and xanthan gum (4 mg) and the resultant mixture may be blended, passedthrough a No. 10 mesh U.S. sieve, and then mixed with a previously madesolution of microcrystalline cellulose and sodium carboxymethylcellulose (11:89, 50 mg) in water. Sodium benzoate (10 mg), flavour, andcolour may be diluted with water and added with stirring. Sufficientwater may then be added with stirring. Further sufficient water may bethen added to produce a total volume of 5 mL.

Formulation 4—Tablets

A salt of the invention may be admixed as a dry powder with a drygelatin binder in an approximate 1:2 weight ratio. A minor amount oflubritab may be added as a lubricant. The mixture may be formed into25-900 mg tablets (8-300 mg of active salt of the invention) in a tabletpress.

Formulation 5—Injection

A salt of the invention may be dissolved or suspended in a bufferedsterile saline injectable aqueous medium to a concentration ofapproximately 5 mg/mL.

Formulation 6—Topical

Stearyl alcohol (250 g) and a white petrolatum (250 g) may be melted atabout 75° C. and then a mixture of A salt of the invention (50 g)methylparaben (0.25 g), propylparaben (0.15 g), sodium lauryl sulfate(10 g), and propylene glycol (120 g) dissolved in water (about 370 g)may be added and the resulting mixture may be stirred until it congeals.

Methods of Treatment

In one embodiment, the present invention provides salts of theinvention, or pharmaceutical compositions comprising a salt of theinvention, for use in medicine. In a particular embodiment, the presentinvention provides salts of the invention or pharmaceutical compositionscomprising a salt of the invention, for use in the prophylaxis and/ortreatment of inflammatory conditions, autoimmune diseases, proliferativediseases, allergy, transplant rejection, diseases involving degradationand/or disruption of cartilage homeostasis, congenital cartilagemalformations, and/or diseases associated with hypersecretion of IL6 orinterferons.

In one embodiment, the present invention provides the use of a salt ofthe invention, or pharmaceutical compositions comprising a salt of theinvention in medicine. In a particular embodiment, the present inventionprovides salts of the invention or pharmaceutical compositionscomprising a salt of the invention, for use in the prophylaxis and/ortreatment of inflammatory conditions, autoimmune diseases, proliferativediseases, allergy, transplant rejection, diseases involving degradationand/or disruption of cartilage homeostasis, congenital cartilagemalformations, and/or diseases associated with hypersecretion of IL6 orinterferons.

In another embodiment, the present invention provides salts of theinvention, or pharmaceutical compositions comprising a salt of theinvention for use in the manufacture of a medicament for use in theprophylaxis and/or treatment of inflammatory conditions, autoimmunediseases, proliferative diseases, allergy, transplant rejection,diseases involving degradation and/or disruption of cartilagehomeostasis, congenital cartilage malformations, and/or diseasesassociated with hypersecretion of IL6 or interferons.

In additional method of treatment aspects, this invention providesmethods of prophylaxis and/or treatment of a mammal afflicted withinflammatory conditions, autoimmune diseases, proliferative diseases,allergy, transplant rejection, diseases involving degradation and/ordisruption of cartilage homeostasis, congenital cartilage malformations,and/or diseases associated with hypersecretion of IL6 or interferons,which methods comprise the administration of an effective amount of asalt of the invention or one or more of the pharmaceutical compositionsherein described for the treatment or prophylaxis of said condition.

In one embodiment, the present invention provides pharmaceuticalcompositions comprising a salt of the invention, and another therapeuticagent. In a particular embodiment, the other therapeutic agent is anagent for the treatment of inflammatory conditions, autoimmune diseases,proliferative diseases, allergy, transplant rejection, diseasesinvolving degradation and/or disruption of cartilage homeostasis,congenital cartilage malformations, and/or diseases associated withhypersecretion of IL6 or interferons.

In one embodiment, the present invention provides salts of the inventionor pharmaceutical compositions comprising a salt of the invention, foruse in the prophylaxis and/or treatment of inflammatory diseases. In aparticular embodiment, the inflammatory disease is selected fromrheumatoid arthritis, osteoarthritis, allergic airway disease (e.g.asthma), chronic obstructive pulmonary disease (COPD) and inflammatorybowel diseases (e.g. Crohn's disease, Whipple, chronic ulcerativecolitis, or colitis). More particularly, the inflammatory disease isselected from rheumatoid arthritis, and inflammatory bowel diseases(e.g. Crohn's disease, Whipple, chronic ulcerative colitis, or colitis).

In one embodiment, the present invention provides the use of a salt ofthe invention or pharmaceutical compositions comprising a salt of theinvention, in the prophylaxis and/or treatment of inflammatory diseases.In a particular embodiment, the inflammatory disease is selected fromrheumatoid arthritis, osteoarthritis, allergic airway disease (e.g.asthma), chronic obstructive pulmonary disease (COPD) and inflammatorybowel diseases (e.g. Crohn's disease, Whipple, chronic ulcerativecolitis, or colitis). More particularly, the inflammatory disease isselected from rheumatoid arthritis, and inflammatory bowel diseases(e.g. Crohn's disease, Whipple, chronic ulcerative colitis, or colitis).

In another embodiment, the present invention provides salts of theinvention, or pharmaceutical compositions comprising a salt of theinvention for use in the manufacture of a medicament for use in theprophylaxis and/or treatment of inflammatory diseases. In a particularembodiment, the inflammatory disease is selected from rheumatoidarthritis, osteoarthritis, allergic airway disease (e.g. asthma),chronic obstructive pulmonary disease (COPD) and inflammatory boweldiseases (e.g. Crohn's disease, Whipple, chronic ulcerative colitis, orcolitis). More particularly, the inflammatory disease is selected fromrheumatoid arthritis, and inflammatory bowel diseases (e.g. Crohn'sdisease, Whipple, chronic ulcerative colitis, or colitis).

In additional method of treatment aspects, this invention providesmethods of prophylaxis and/or treatment of a mammal afflicted withinflammatory diseases, which methods comprise the administration of aneffective amount of a salt of the invention or one or more of thepharmaceutical compositions herein described for the treatment orprophylaxis of said condition. In a particular embodiment, theinflammatory disease is selected from rheumatoid arthritis,osteoarthritis, allergic airway disease (e.g. asthma), chronicobstructive pulmonary disease (COPD) and inflammatory bowel diseases(e.g. Crohn's disease, Whipple, chronic ulcerative colitis, or colitis).More particularly, the inflammatory disease is selected from rheumatoidarthritis, and inflammatory bowel diseases (e.g. Crohn's disease,Whipple, chronic ulcerative colitis, or colitis).

In one embodiment, the present invention provides salts of the inventionor pharmaceutical compositions comprising a salt of the invention, foruse in the prophylaxis and/or treatment of autoimmune diseases. In aparticular embodiment, the autoimmune disease is selected from COPD,asthma (e.g. intrinsic asthma, extrinsic asthma, dust asthma, infantileasthma) particularly chronic or inveterate asthma (for example lateasthma and airway hyperresponsiveness), bronchitis, including bronchialasthma, systemic lupus crythematosus (SLE), cutaneous lupuscrythematosus, lupus nephritis, dermatomyositis, Sjogren's syndrome,multiple sclerosis, psoriasis, dry eye disease, type I diabetes mellitusand complications associated therewith, atopic eczema (atopicdermatitis), thyroiditis (Hashimoto's and autoimmune thyroiditis),contact dermatitis and further eczematous dermatitis, inflammatory boweldisease (e.g. Crohn's disease, Whipple, chronic ulcerative colitis, orcolitis), atherosclerosis and amyotrophic lateral sclerosis.Particularly, the autoimmune disease is selected from COPD, asthma,systemic lupus erythematosus, type I diabetes mellitus and inflammatorybowel disease.

In one embodiment, the present invention provides the use of a salt ofthe invention or pharmaceutical compositions comprising a salt of theinvention, in the prophylaxis and/or treatment of autoimmune diseases.In a particular embodiment, the autoimmune disease is selected fromCOPD, asthma (e.g. intrinsic asthma, extrinsic asthma, dust asthma,infantile asthma) particularly chronic or inveterate asthma (for examplelate asthma and airway hyperresponsiveness), bronchitis, includingbronchial asthma, systemic lupus erythematosus (SLE), cutaneous lupuserythematosus, lupus nephritis, dermatomyositis, Sjogren's syndrome,multiple sclerosis, psoriasis, dry eye disease, type I diabetes mellitusand complications associated therewith, atopic eczema (atopicdermatitis), thyroiditis (Hashimoto's and autoimmune thyroiditis),contact dermatitis and further eczematous dermatitis, inflammatory boweldisease (e.g. Crohn's disease, Whipple, chronic ulcerative colitis, orcolitis), atherosclerosis and amyotrophic lateral sclerosis.Particularly, the autoimmune disease is selected from COPD, asthma,systemic lupus erythematosus, type I diabetes mellitus and inflammatorybowel disease.

In another embodiment, the present invention provides salts of theinvention, or pharmaceutical compositions comprising a salt of theinvention for use in the manufacture of a medicament for use in theprophylaxis and/or treatment of autoimmune diseases. In a particularembodiment, the autoimmune disease is selected from COPD, asthma (e.g.intrinsic asthma, extrinsic asthma, dust asthma, infantile asthma)particularly chronic or inveterate asthma (for example late asthma andairway hyperresponsiveness), bronchitis, including bronchial asthma,systemic lupus erythematosus (SLE), cutaneous lupus erythematosus, lupusnephritis, dermatomyositis, Sjogren's syndrome, multiple sclerosis,psoriasis, dry eye disease, type I diabetes mellitus and complicationsassociated therewith, atopic eczema (atopic dermatitis), thyroiditis(Hashimoto's and autoimmune thyroiditis), contact dermatitis and furthereczematous dermatitis, inflammatory bowel disease (e.g. Crohn's disease,Whipple, chronic ulcerative colitis, or colitis), atherosclerosis andamyotrophic lateral sclerosis. Particularly, the autoimmune disease isselected from COPD, asthma, systemic lupus erythematosus, type Idiabetes mellitus and inflammatory bowel disease.

In additional method of treatment aspects, this invention providesmethods of prophylaxis and/or treatment of a mammal afflicted withautoimmune diseases, which methods comprise the administration of aneffective amount of a salt of the invention or one or more of thepharmaceutical compositions herein described for the treatment orprophylaxis of said condition. In a particular embodiment, theautoimmune disease is selected from COPD, asthma (e.g. intrinsic asthma,extrinsic asthma, dust asthma, infantile asthma) particularly chronic orinveterate asthma (for example late asthma and airwayhyperreponsiveness), bronchitis, including bronchial asthma, systemiclupus erythematosus (SLE), cutaneous lupus erythematosus, lupusnephritis, dermatomyositis, Sjogren's syndrome, multiple sclerosis,psoriasis, dry eye disease, type I diabetes mellitus and complicationsassociated therewith, atopic eczema (atopic dermatitis), thyroiditis(Hashimoto's and autoimmune thyroiditis), contact dermatitis and furthereczematous dermatitis, inflammatory bowel disease (e.g. Crohn's disease,Whipple, chronic ulcerative colitis, or colitis), atherosclerosis andamyotrophic lateral sclerosis. Particularly, the autoimmune disease isselected from COPD, asthma, systemic lupus erythematosus, type Idiabetes mellitus and inflammatory bowel disease.

In one embodiment, the present invention provides salts of the inventionor pharmaceutical compositions comprising a salt of the invention, foruse in the prophylaxis and/or treatment of proliferative diseases. In aparticular embodiment, the proliferative disease is selected fromcancer, and leukaemia. In a more particular embodiment, theproliferative disease is selected from breast cancer, endometrial andcervical cancer, lung cancer, ovarian cancer, prostate cancer, hepaticcancer, and pancreatic cancer.

In one embodiment, the present invention provides the use of a salt ofthe invention or pharmaceutical compositions comprising a salt of theinvention, in the prophylaxis and/or treatment of proliferativediseases. In a particular embodiment, the proliferative disease isselected from cancer, and leukaemia. In a more particular embodiment,the proliferative disease is selected from breast cancer, endometrialand cervical cancer, lung cancer, ovarian cancer, prostate cancer,hepatic cancer, and pancreatic cancer.

In another embodiment, the present invention provides salts of theinvention, or pharmaceutical compositions comprising a salt of theinvention for use in the manufacture of a medicament for use in theprophylaxis and/or treatment of proliferative diseases. In a particularembodiment, the proliferative disease is selected from cancer, andleukaemia. In a more particular embodiment, the proliferative disease isselected from breast cancer, endometrial and cervical cancer, lungcancer, ovarian cancer, prostate cancer, hepatic cancer, and pancreaticcancer.

In additional method of treatment aspects, this invention providesmethods of prophylaxis and/or treatment of a mammal afflicted withproliferative diseases, which methods comprise the administration of aneffective amount of a salt of the invention or one or more of thepharmaceutical compositions herein described for the treatment orprophylaxis of said condition. In a particular embodiment, theproliferative disease is selected from cancer, and leukaemia. In a moreparticular embodiment, the proliferative disease is selected from breastcancer, endometrial and cervical cancer, lung cancer, ovarian cancer,prostate cancer, hepatic cancer, and pancreatic cancer.

In one embodiment, the present invention provides salts of the inventionor pharmaceutical compositions comprising a salt of the invention, foruse in the prophylaxis and/or treatment of allergy. In a particularembodiment, the allergy is selected from allergic airway disease (e.g.asthma, rhinitis), sinusitis, eczema and hives, as well as foodallergies or allergies to insect venom.

In one embodiment, the present invention provides the use of a salt ofthe invention or pharmaceutical compositions comprising a salt of theinvention, in the prophylaxis and/or treatment of allergy. In aparticular embodiment, the allergy is selected from allergic airwaydisease (e.g. asthma, rhinitis), sinusitis, eczema and hives, as well asfood allergies or allergies to insect venom.

In another embodiment, the present invention provides salts of theinvention, or pharmaceutical compositions comprising a salt of theinvention for use in the manufacture of a medicament for use in theprophylaxis and/or treatment of allergy. In a particular embodiment, theallergy is selected from allergic airway disease (e.g. asthma,rhinitis), sinusitis, eczema and hives, as well as food allergies orallergies to insect venom.

In additional method of treatment aspects, this invention providesmethods of prophylaxis and/or treatment of a mammal afflicted withallergy, which methods comprise the administration of an effectiveamount of a salt of the invention or one or more of the pharmaceuticalcompositions herein described for the treatment or prophylaxis of saidcondition. In a particular embodiment, the allergy is selected fromallergic airway disease (e.g. asthma, rhinitis), sinusitis, eczema andhives, as well as food allergies or allergies to insect venom.

In one embodiment, the present invention provides salts of the inventionor pharmaceutical compositions comprising a salt of the invention, foruse in the prophylaxis and/or treatment of transplant rejection.

In one embodiment, the present invention provides the use of a salt ofthe invention or pharmaceutical compositions comprising a salt of theinvention, in the prophylaxis and/or treatment of transplant rejection.

In another embodiment, the present invention provides salts of theinvention, or pharmaceutical compositions comprising a salt of theinvention for use in the manufacture of a medicament for use in theprophylaxis and/or treatment of transplant rejection.

In additional method of treatment aspects, this invention providesmethods of prophylaxis and/or treatment of a mammal afflicted withtransplant rejection, which methods comprise the administration of aneffective amount of a salt of the invention or one or more of thepharmaceutical compositions herein described for the treatment orprophylaxis of said condition.

In one embodiment, the present invention provides salts of the inventionor pharmaceutical compositions comprising a salt of the invention, foruse in the prophylaxis and/or treatment of diseases involvingdegradation and/or disruption of cartilage homeostasis. In a particularembodiment, the diseases involving degradation and/or disruption ofcartilage homeostasis is selected from osteoarthritis, psoriaticarthritis, juvenile rheumatoid arthritis, gouty arthritis, septic orinfectious arthritis, reactive arthritis, reflex sympathetic dystrophy,algodystrophy, achondroplasia, Paget's disease, Tietze syndrome orcostal chondritis, fibromyalgia, osteochondritis, neurogenic orneuropathic arthritis, arthropathy, sarcoidosis, amylosis, hydarthrosis,periodical disease, rheumatoid spondylitis, endemic forms of arthritislike osteoarthritis deformans endemica, Mseleni disease and Handigodudisease; degeneration resulting from fibromyalgia, systemic lupuserythematosus, scleroderma and ankylosing spondylitis.

In one embodiment, the present invention provides the use of a salt ofthe invention or pharmaceutical compositions comprising a salt of theinvention, in the prophylaxis and/or treatment of diseases involvingdegradation and/or disruption of cartilage homeostasis. In a particularembodiment, the diseases involving degradation and/or disruption ofcartilage homeostasis is selected from osteoarthritis, psoriaticarthritis, juvenile rheumatoid arthritis, gouty arthritis, septic orinfectious arthritis, reactive arthritis, reflex sympathetic dystrophy,algodystrophy, achondroplasia, Paget's disease, Tietze syndrome orcostal chondritis, fibromyalgia, osteochondritis, neurogenic orneuropathic arthritis, arthropathy, sarcoidosis, amylosis, hydarthrosis,periodical disease, rheumatoid spondylitis, endemic forms of arthritislike osteoarthritis deformans endemica, Mseleni disease and Handigodudisease; degeneration resulting from fibromyalgia, systemic lupuserythematosus, scleroderma and ankylosing spondylitis.

In another embodiment, the present invention provides salts of theinvention, or pharmaceutical compositions comprising a salt of theinvention for use in the manufacture of a medicament for use in theprophylaxis and/or treatment of diseases involving degradation and/ordisruption of cartilage homeostasis. In a particular embodiment, thediseases involving degradation and/or disruption of cartilagehomeostasis is selected from osteoarthritis, psoriatic arthritis,juvenile rheumatoid arthritis, gouty arthritis, septic or infectiousarthritis, reactive arthritis, reflex sympathetic dystrophy,algodystrophy, achondroplasia, Paget's disease, Tietze syndrome orcostal chondritis, fibromyalgia, osteochondritis, neurogenic orneuropathic arthritis, arthropathy, sarcoidosis, amylosis, hydarthrosis,periodical disease, rheumatoid spondylitis, endemic forms of arthritislike osteoarthritis deformans endemica, Mseleni disease and Handigodudisease; degeneration resulting from fibromyalgia, systemic lupuserythematosus, scleroderma and ankylosing spondylitis.

In additional method of treatment aspects, this invention providesmethods of prophylaxis and/or treatment of a mammal afflicted withdiseases involving degradation and/or disruption of cartilagehomeostasis, which methods comprise the administration of an effectiveamount of a salt of the invention or one or more of the pharmaceuticalcompositions herein described for the treatment or prophylaxis of saidcondition. In a particular embodiment, the diseases involvingdegradation and/or disruption of cartilage homeostasis is selected fromosteoarthritis, psoriatic arthritis, juvenile rheumatoid arthritis,gouty arthritis, septic or infectious arthritis, reactive arthritis,reflex sympathetic dystrophy, algodystrophy, achondroplasia, Paget'sdisease, Tietze syndrome or costal chondritis, fibromyalgia,osteochondritis, neurogenic or neuropathic arthritis, arthropathy,sarcoidosis, amylosis, hydarthrosis, periodical disease, rheumatoidspondylitis, endemic forms of arthritis like osteoarthritis deformansendemica, Mseleni disease and Handigodu disease; degeneration resultingfrom fibromyalgia, systemic lupus erythematosus, scleroderma andankylosing spondylitis.

In one embodiment, the present invention provides salts of the inventionor pharmaceutical compositions comprising a salt of the invention, foruse in the prophylaxis and/or treatment of congenital cartilagemalformation(s). In a particular embodiment, the congenital cartilagemalformation(s) is selected from hereditary chondrolysis,chondrodysplasias and pseudochondrodysplasias, in particular, butwithout limitation, microtia, anotia, metaphyseal chondrodysplasia, andrelated disorders.

In one embodiment, the present invention provides the use of a salt ofthe invention or pharmaceutical compositions comprising a salt of theinvention, in the prophylaxis and/or treatment of congenital cartilagemalformation(s). In a particular embodiment, the congenital cartilagemalformation(s) is selected from hereditary chondrolysis,chondrodysplasias and pseudochondrodysplasias, in particular, butwithout limitation, microtia, anotia, metaphyseal chondrodysplasia, andrelated disorders.

In another embodiment, the present invention provides salts of theinvention, or pharmaceutical compositions comprising a salt of theinvention for use in the manufacture of a medicament for use in theprophylaxis and/or treatment of congenital cartilage malformation(s). Ina particular embodiment, the congenital cartilage malformation(s) isselected from hereditary chondrolysis, chondrodysplasias andpseudochondrodysplasias, in particular, but without limitation,microtia, anotia, metaphyseal chondrodysplasia, and related disorders.

In additional method of treatment aspects, this invention providesmethods of prophylaxis and/or treatment of a mammal afflicted withcongenital cartilage malformation(s), which methods comprise theadministration of an effective amount of a salt of the invention or oneor more of the pharmaceutical compositions herein described for thetreatment or prophylaxis of said condition. In a particular embodiment,the congenital cartilage malformation(s) is selected from hereditarychondrolysis, chondrodysplasias and pseudochondrodysplasias, inparticular, but without limitation, microtia, anotia, metaphysealchondrodysplasia, and related disorders.

In one embodiment, the present invention provides salts of the inventionor pharmaceutical compositions comprising a salt of the invention, foruse in the prophylaxis and/or treatment of disease(s) associated withhypersecretion of IL6. In a particular embodiment, the disease(s)associated with hypersecretion of IL6 is selected from Castleman'sdisease, multiple myeloma, psoriasis, Kaposi's sarcoma and/or mesangialproliferative glomerulonephritis.

In one embodiment, the present invention provides the use of a salt ofthe invention or pharmaceutical compositions comprising a salt of theinvention, in the prophylaxis and/or treatment of disease(s) associatedwith hypersecretion of IL6. In a particular embodiment, the disease(s)associated with hypersecretion of IL6 is selected from Castleman'sdisease, multiple myeloma, psoriasis, Kaposi's sarcoma and/or mesangialproliferative glomerulonephritis.

In another embodiment, the present invention provides salts of theinvention, or pharmaceutical compositions comprising a salt of theinvention for use in the manufacture of a medicament for use in theprophylaxis and/or treatment of disease(s) associated withhypersecretion of IL6. In a particular embodiment, the disease(s)associated with hypersecretion of IL6 is selected from Castleman'sdisease, multiple myeloma, psoriasis, Kaposi's sarcoma and/or mesangialproliferative glomerulonephritis.

In additional method of treatment aspects, this invention providesmethods of prophylaxis and/or treatment of a mammal afflicted withdisease(s) associated with hypersecretion of IL6, which methods comprisethe administration of an effective amount of a salt of the invention orone or more of the pharmaceutical compositions herein described for thetreatment or prophylaxis of said condition. In a particular embodiment,the disease(s) associated with hypersecretion of IL6 is selected fromCastleman's disease, multiple myeloma, psoriasis, Kaposi's sarcomaand/or mesangial proliferative glomerulonephritis.

In one embodiment, the present invention provides salts of the inventionor pharmaceutical compositions comprising a salt of the invention, foruse in the prophylaxis and/or treatment of disease(s) associated withhypersecretion of interferons. In a particular embodiment, the diseaseassociated with hypersecretion of interferons is selected from systemicand cutaneous lupus erythematosus, lupus nephritis, dermatomyositis,Sjogren's syndrome, psoriasis, and rheumatoid arthritis.

In one embodiment, the present invention provides the use of a salt ofthe invention or pharmaceutical compositions comprising a salt of theinvention, in the prophylaxis and/or treatment of disease(s) associatedwith hypersecretion of interferons. In a particular embodiment, thedisease associated with hypersecretion of interferons is selected fromsystemic and cutaneous lupus erythematosus, lupus nephritis,dermatomyositis, Sjogren's syndrome, psoriasis, and rheumatoidarthritis.

In another embodiment, the present invention provides salts of theinvention, or pharmaceutical compositions comprising a salt of theinvention for use in the manufacture of a medicament for use in theprophylaxis and/or treatment of disease(s) associated withhypersecretion of interferons. In a particular embodiment, the diseaseassociated with hypersecretion of interferons is selected from systemicand cutaneous lupus erythematosus, lupus nephritis, dermatomyositis,Sjogren's syndrome, psoriasis, and rheumatoid arthritis.

In additional method of treatment aspects, this invention providesmethods of prophylaxis and/or treatment of a mammal afflicted withdisease(s) associated with hypersecretion of interferons, which methodscomprise the administration of an effective amount of a salt of theinvention or one or more of the pharmaceutical compositions hereindescribed for the treatment or prophylaxis of said condition. In aparticular embodiment, the disease associated with hypersecretion ofinterferons is selected from systemic and cutaneous lupus erythematosus,lupus nephritis, dermatomyositis, Sjogren's syndrome, psoriasis, andrheumatoid arthritis.

A particular regimen of the present method comprises the administrationto a subject suffering from inflammatory conditions, autoimmunediseases, proliferative diseases, allergy, transplant rejection,diseases involving degradation and/or disruption of cartilagehomeostasis, congenital cartilage malformations, and/or diseasesassociated with hypersecretion of IL6 or interferons, of an effectiveamount of a salt of the invention for a period of time sufficient toreduce the level of the aforementioned diseases in the subject, andpreferably terminate the processes responsible for said diseases.

Using these dosing patterns, each dose provides from about 1 to about500 mg of the salt of the invention, with particular doses eachproviding from about 10 to about 300 mg, more particularly about 25 toabout 250 mg, and especially 10 mg, 20 mg, 25 mg, 50 mg, 75 mg, 100 mg,150 mg, 200 mg, 250 mg, or 300 mg.

Injection dose levels range from about 0.1 mg/kg/h to at least 10mg/kg/h, all for from about 1 to about 120 h and especially 24 to 96 h.A preloading bolus of from about 0.1 mg/kg to about 10 mg/kg or more mayalso be administered to achieve adequate steady state levels. Themaximum total dose is not expected to exceed about 1 g/day for a 40 to80 kg human patient.

For the prophylaxis and/or treatment of long-term conditions, such asdegenerative conditions, the regimen for treatment usually stretchesover many months or years so oral dosing is preferred for patientconvenience and tolerance. With oral dosing, one to four (1-4) regulardoses daily, especially one to three (1-3) regular doses daily,typically one to two (1-2) regular doses daily, and most typically one(1) regular dose daily are representative regimens. Alternatively forlong lasting effect drugs, with oral dosing, once every other week, onceweekly, and once a day are representative regimens. In particular,dosage regimen can be every 1-14 days, more particularly 1-10 days, evenmore particularly 1-7 days, and most particularly 1-3 days.

Using these dosing patterns, each dose provides from about 1 to about500 mg of the salt of the invention, with particular doses eachproviding from about 10 to about 300 mg, more particularly about 25 toabout 250 mg, and especially 10 mg, 20 mg, 25 mg, 50 mg, 75 mg, 100 mg,150 mg, 200 mg, 250 mg, or 300 mg.

Transdermal doses are generally selected to provide similar or lowerblood levels than are achieved using injection doses.

When used to prevent the onset of a condition, a salt of the inventionwill be administered to a patient at risk for developing the condition,typically on the advice and under the supervision of a physician, at thedosage levels described above. Patients at risk for developing aparticular condition generally include those that have a family historyof the condition, or those who have been identified by genetic testingor screening to be particularly susceptible to developing the condition.

A salt of the invention can be administered as the sole active agent orit can be administered in combination with other therapeutic agents,including other salt of the inventions that demonstrate the same or asimilar therapeutic activity and that are determined to be safe andefficacious for such combined administration. In a specific embodiment,co-administration of two (or more) agents allows for significantly lowerdoses of each to be used, thereby reducing the side effects seen.

In one embodiment, a salt of the invention or a pharmaceuticalcomposition comprising a salt of the invention is administered as amedicament. In a specific embodiment, said pharmaceutical compositionadditionally comprises a further active ingredient.

In one embodiment, a salt of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis of adisease involving inflammation, particular agents include, but are notlimited to, immunoregulatory agents e.g. azathioprine, corticosteroids(e.g. prednisolone or dexamethasone), cyclophosphamide, cyclosporin A,tacrolimus, mycophenolate, mofetil, muromonab-CD3 (OKT3, e.g.Orthocolone®), ATG, aspirin, acetaminophen, ibuprofen, naproxen, andpiroxicam.

In one embodiment, a salt of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis ofarthritis (e.g. rheumatoid arthritis), particular agents include but arenot limited to analgesics, non-steroidal anti-inflammatory drugs(NSAIDS), steroids, synthetic DMARDS (for example but without limitationmethotrexate, leflunomide, sulfasalazine, auranofin, sodiumaurothiomalate, penicillamine, chloroquine, hydroxychloroquine,azathioprine, tofacitinib, baricitinib, fostamatinib, and cyclosporin),and biological DMARDS (for example but without limitation infliximab,etanercept, adalimumab, rituximab, and abatacept).

In one embodiment, a salt of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis ofproliferative disorders, particular agents include but are not limitedto: methotrexate, leukovorin, adriamycin, prednisone, bleomycin,cyclophosphamide, 5-fluorouracil, paclitaxel, docetaxel, vincristine,vinblastine, vinorelbine, doxorubicin, tamoxifen, toremifene, megestrolacetate, anastrozole, goserelin, anti-HER2 monoclonal antibody (e.g.Herceptin™) capecitabine, raloxifene hydrochloride, EGFR inhibitors(e.g. Iressa®, Tarceva™, Erbitux™), VEGF inhibitors (e.g. Avastin™),proteasome inhibitors (e.g. Velcade™), Glivec® and hsp90 inhibitors(e.g. 17-AAG). Additionally, the salt of the invention according toFormula I may be administered in combination with other therapiesincluding, but not limited to, radiotherapy or surgery. In a specificembodiment the proliferative disorder is selected from cancer,myeloproliferative disease or leukaemia.

In one embodiment, a salt of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis ofautoimmune diseases, particular agents include but are not limited to:glucocorticoids, cytostatic agents (e.g. purine analogs), alkylatingagents, (e.g. nitrogen mustards (cyclophosphamide), nitrosoureas,platinum salt of the inventions, and others), antimetabolites (e.g.methotrexate, azathioprine and mercaptopurine), cytotoxic antibiotics(e.g. dactinomycin anthracyclines, mitomycin C, blcomycin, andmithramycin), antibodies (e.g. anti-CD20, anti-CD25 or anti-CD3 (OTK3)monoclonal antibodies, Atgam® and Thymoglobuline®), cyclosporin,tacrolimus, rapamycin (sirolimus), interferons (e.g. IFN-β), TNF bindingproteins (e.g. infliximab, etanercept, or adalimumab), mycophenolate,fingolimod and myriocin.

In one embodiment, a salt of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis oftransplant rejection, particular agents include but are not limited to:calcineurin inhibitors (e.g. cyclosporin or tacrolimus (FK506)), mTORinhibitors (e.g. sirolimus, everolimus), anti-proliferatives (e.g.azathioprine, mycophenolic acid), corticosteroids (e.g. prednisolone,hydrocortisone), antibodies (e.g. monoclonal anti-IL-2Rα receptorantibodies, basiliximab, daclizumab), polyclonal anti-T-cell antibodies(e.g. anti-thymocyte globulin (ATG), anti-lymphocyte globulin (ALG)).

In one embodiment, a salt of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis of asthmaand/or rhinitis and/or COPD, particular agents include but are notlimited to: beta2-adrenoceptor agonists (e.g. salbutamol, levalbuterol,terbutaline and bitolterol), epinephrine (inhaled or tablets),anticholinergics (e.g. ipratropium bromide), glucocorticoids (oral orinhaled). Long-acting β2-agonists (e.g. salmeterol, formoterol,bambuterol, and sustained-release oral albuterol), combinations ofinhaled steroids and long-acting bronchodilators (e.g.fluticasone/salmeterol, budesonide/formotcrol), leukotriene antagonistsand synthesis inhibitors (e.g. montclukast, zafirlukast and zileuton),inhibitors of mediator release (e.g. cromoglycate and ketotifen),biological regulators of IgE response (e.g. omalizumab), antihistamines(e.g. ceterizine, cinnarizine, fexofenadine) and vasoconstrictors (e.g.oxymethazoline, xylomethazoline, nafazoline and tramazoline).

Additionally, a salt of the invention may be administered in combinationwith emergency therapies for asthma and/or COPD, such therapies includeoxygen or heliox administration, nebulized salbutamol or terbutaline(optionally combined with an anticholinergic (e.g. ipratropium),systemic steroids (oral or intravenous, e.g. prednisone, prednisolone,methylprednisolone, dexamethasone, or hydrocortisone), intravenoussalbutamol, non-specific beta-agonists, injected or inhaled (e.g.epinephrine, isoetharine, isoproterenol, metaproterenol),anticholinergics (IV or nebulized, e.g. glycopyrrolate, atropine,ipratropium), methylxanthines (theophylline, aminophylline,bamiphylline), inhalation anesthetics that have a bronchodilatory effect(e.g. isoflurane, halothane, enflurane), ketamine and intravenousmagnesium sulfate.

In one embodiment, a salt of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis ofinflammatory bowel disease (IBD), particular agents include but are notlimited to: glucocorticoids (e.g. prednisone, budesonide) syntheticdisease modifying, immunomodulatory agents (e.g. methotrexate,leflunomide, sulfasalazine, mesalazine, azathioprine, 6-mercaptopurineand cyclosporin) and biological disease modifying, immunomodulatoryagents (infliximab, adalimumab, rituximab, and abatacept).

In one embodiment, a salt of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis of SLE,particular agents include but are not limited to: human monoclonalantibodies (belimumab (Benlysta)), Disease-modifying antirheumatic drugs(DMARDs) such as antimalarials (e.g. plaquenil, hydroxychloroquine),immunosuppressants (e.g. methotrexate and azathioprine),cyclophosphamide and mycophenolic acid, immunosuppressive drugs andanalgesics, such as nonsteroidal anti-inflammatory drugs, opiates (e.g.dextropropoxyphene and co-codamol), opioids (e.g. hydrocodone,oxycodone, MS Contin, or methadone) and the fentanyl duragesictransdermal patch.

In one embodiment, a salt of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis ofpsoriasis, particular agents include but are not limited to: topicaltreatments such as bath solutions, moisturizers, medicated creams andointments containing coal tar, dithranol (anthralin), corticosteroidslike desoximetasone (Topicort™), fluocinonide, vitamin D3 analogues (forexample, calcipotriol), argan oil and retinoids (etretinate, acitretin,tazarotene), systemic treatments such as methotrexate, cyclosporine,retinoids, tioguanine, hydroxyurea, sulfasalazine, mycophenolatemofetil, azathioprine, tacrolimus, fumaric acid esters or biologics suchas Amevive™ Enbrel™, Humira™, Remicade™, Raptiva™ and ustekinumab (a IL12 and IL-23 blocker). Additionally, a salt of the invention may beadministered in combination with other therapies including, but notlimited to phototherapy, or photochemotherapy (e.g. psoralen andultraviolet A phototherapy (PUVA)).

In one embodiment, a salt of the invention is co-administered withanother therapeutic agent for the treatment and/or prophylaxis ofallergic reaction, particular agents include but are not limited to:antihistamines (e.g. cetirizine, diphenhydramine, fexofenadine,levocetirizine), glucocorticoids (e.g. prednisone, betamethasone,beclomethasone, dexamethasone), epinephrine, theophylline oranti-leukotrienes (e.g. montelukast or zafirlukast), anti-cholinergicsand decongestants.

By co-administration is included any means of delivering two or moretherapeutic agents to the patient as part of the same treatment regime,as will be apparent to the skilled person. Whilst the two or more agentsmay be administered simultaneously in a single formulation, i.e. as asingle pharmaceutical composition, this is not essential. The agents maybe administered in different formulations and at different times.

Chemical Synthetic Procedures General

The compound according to Formula I, and the salts of the invention canbe prepared from readily available starting materials using thefollowing general methods and procedures. It will be appreciated thatwhere typical or preferred process conditions (i.e. reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. Optimum reaction conditions may vary with theparticular reactants or solvent used, but such conditions can bedetermined by one skilled in the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. The choice of asuitable protecting group for a particular functional group as well assuitable conditions for protection and deprotection are well known inthe art (Greene, T W; Wuts, P G M; 1991).

The following methods are presented with details as to the preparationof a salt of the invention as defined hereinabove and the comparativeexamples. A salt of the invention may be prepared from known orcommercially available starting materials and reagents by one skilled inthe art of organic synthesis.

All reagents are of commercial grade and are used as received withoutfurther purification, unless otherwise stated. Commercially availableanhydrous solvents are used for reactions conducted under inertatmosphere. Reagent grade solvents are used in all other cases, unlessotherwise specified. Column chromatography is performed on silica gel 60(35-70 μm). Thin layer chromatography is carried out using pre-coatedsilica gel F-254 plates (thickness 0.25 mm). ¹H NMR spectra are recordedon a Bruker DPX 400 NMR spectrometer (400 MHz or a Bruker Advance 300NMR spectrometer (300 MHz). Chemical shifts (δ) for 1H NMR spectra arereported in parts per million (ppm) relative to tetramethylsilane (δ0.00) or the appropriate residual solvent peak, i.e. CHCl₃ (δ 7.27), asinternal reference. Multiplicities are given as singlet (s), doublet(d), triplet (t), quartet (q), quintuplet (quin), multiplet (m) andbroad (br). Electrospray MS spectra are obtained on a Waters platformLC/MS spectrometer or with Waters Acquity H-Class UPLC coupled to aWaters Mass detector 3100 spectrometer. Columns used: Waters AcquityUPLC BEH C18 1.7 μm, 2.1 mm ID×50 mm L, Waters Acquity UPLC BEH C18 1.7μm, 2.1 mm ID×30 mm L, or Waters Xterra MS 5 μm C18, 100×4.6 mm. Themethods are using either MeCN/H₂O gradients (H₂O contains either 0.1%TFA or 0.1% NH₃) or MeOH/H₂O gradients (H₂O contains 0.05% TFA).Microwave heating is performed with a Biotage Initiator.

TABLE I List of abbreviations used in the experimental section:Abbreviation Definition APMA 4-aminophenylmercuric acetate app tApparent triplet ATP Adenosine-5′-triphosphate AUC Area Under the Curvebd Broad doublet bs Broad singlet BSA Bovine serum albumine bt Broadtriplet Cat. Catalytic amount cDNA copy deoxyribonucleic acid d doubletDCM Dichloromethane Desc′d Described in details DLM Data Logger ModuleDMSO Dimethylsulfoxide DSC Differential scanning calorimetry DTTDithiothreitol DVS Dynamic vapor sorption EDTAEthylenediaminetetraacetic acid eq. Equivalent Et₂O Diethyl ether EtOAcEthyl acetate EtOH Ethanol FBS Fetal bovine serum FT-IR Fouriertransformed Infra-red spectroscopy g gram GVS Gravimetric VapourSorption h hour HPLC High pressure liquid chromatography HP-β-CD-2-Hydroxypropyl-beta- cyclodextrin HRP horseradish peroxydase ILInterleukin Int Intermediate kg kilogram L litre LC-MS LiquidChromatography-Mass Spectrometry LPC lysophosphatidylcholine m multipletMeCN Acetonitrile MEK Methyl ethyl ketone MeOH Methanol μL microliter mgmilligram min minute mL millilitre mmol millimoles MMP Matrix MetalloProteinase MS Ms′d Mass measured by LC-MS MW Molecular weight N.A. Notavailable NBS N-Bromosuccinimide nBuOH n-Butanol NMR Nuclear MagneticResonance ONPG Ortho-nitrophényl-β-galactoside Patt Pattern PBFphosphate buffered forrnalin PBS Phosphate buffered saline PCRPolymerase chain reaction Pd(PPh₃)₄ Tetrakis(triphenylphosphine)palladium(0) Pd/C Palladium on Carbon 10% Pd₂(dba)₃Tris(dibenzylideneacetone) dipalladium(0) PdCl₂dppf [1,1′-Bis(diphenylphosphino)ferrocene] dichloropalladium(II) PEG Polyethyleneglycol ppm part-per-million XRPD Powder X-Ray Diffraction q quadrupletQrtPCR quantitative real-time PCR QTL quantitative trait loci rel volRelative volumes RH Relative humidity RNA Ribonucleic acid Rt retentiontime s singlet sept septuplet SS-NMR Solid state Nuclear MagneticResonance SDTA Simultaneous differential thermal analysis t triplet TBMEtButyl methyl ether TEA Triethylamine TFA Trifluoroacetic acid TGAThermogravimetric analysis THF Tetrahydrofuran

TABLE II Salt study apparatus Chemical Purity Purity analysis isperformed on a Waters Acquity Determination by system equipped with adiode array detector and UPLC Micromass ZQ mass spectrometer usingMassLynx software. TGA TGA data are collected on a Mettler TGA/SDTA 851eequipped with a 34 position auto-sampler. The instrument is temperaturecalibrated using certified indium. Typically 5-30 mg of each sample isloaded onto a pre-weighed aluminium crucible and is heated at 10° C./minfrom ambient temperature to 400° C. A nitrogen purge at 50 mL/min ismaintained over the sample. The instrument control and data analysissoftware is STARe v9.10. Thermodynamic Aqueous solubility is determinedby suspending Aqueous sufficient compound in water or buffer to give aSolubility by maximum final concentration of ≧1 mg · mL⁻¹ of HPLC theparent free-form of the compound. Quantitation is made by HPLC withreference to a standard calibration curve. The solubility is calculatedusing the peak areas determined by integration of the peak found at thesame retention time as the principal peak in the standard injection. GVSSorption isotherms are obtained using a SMS DVS Intrinsic moisturesorption analyser, controlled by SMS Analysis Suite software. The sampletemperature is maintained at 25° C. by the instrument controls. Thehumidity is controlled by mixing streams of dry and wet nitrogen, with atotal flow rate of 200 mL/min. The relative humidity is measured by acalibrated Rotronic probe (dynamic range of 0.1-100% % RH), located nearthe sample. The weight change, (mass relaxation) of the sample as afunction of % RH is constantly monitored by the microbalance (accuracy ±0.005 mg). Typically 5-20 mg of sample is placed in a pre- taredstainless steel mesh basket under ambient conditions. The sample isloaded and unloaded at 40% RH and 25° C. (typical room conditions). Amoisture sorption isotherm is performed as outlined below (2 scansgiving 1 complete cycle). The standard isotherm is performed at 25° C.at 10% RH intervals over a 0.5-90% RH range. Polarised Light Samples arestudied on a Leica DLM polarised light Microscopy microscope with adigital video camera for image (PLM) capture. A small amount of eachsample is placed on a glass slide, mounted in immersion oil and coveredwith a glass slip, the individual particles being separated as well aspossible. The sample is viewed with appropriate magnification andpartially polarised light, coupled to a λ false-colour filter. DSCPerkin Elmer DSC 7. Closed Au crucibles, heating rate: 10 or 20° C./min,range: −50° C. to 250° C., or DSC data are collected on a Mettler DSC823e equipped with a 34 position auto-sampler. The instrument iscalibrated for energy and temperature using certified indium. Typically0.5-3 mg of each sample, in a pin-holed aluminium pan, is heated at 10°C./min from 25° C. to 300° C. A nitrogen purge at 50 mL/min ismaintained over the sample. The instrument control and data analysissoftware is STARe v9.10. FT-IR Data are collected on a Nicolet AvatarFT-IR spectrometer with a Smart DurasamplIR accessory and controlled byOmnic software. NMR ¹H and ¹³C Spectra are obtained using a Varian UnityInova 400 NMR spectrometer with a 5 mm inverse triple resonance probeoperating at 400.12 MHz for proton. Samples are prepared in d₆-DMSO,unless otherwise stated. Inverse gated ¹³C NMR spectra are obtainedusing a Bruker DPX300 spectrometer using a dual ¹H/¹³C probe operatingat 75.46 MHz for carbon. The sample is prepared by dissolving ~50 mg ofmaterial in d₆-DMSO. A D1 of thirty seconds is employed with 7168 scans.SS-NMR ¹³C Solid-state NMR spectra are recorded using a Varian VNMRSspectrometer operating at 100.56 MHz for ¹³C and with a 6 mm (outsidediameter) magic-angle spinning (MAS) probe. They are obtained usingcross polarisation and MAS with a 30 seconds recycle delay, 1millisecond contact time and at a sample spin-rate of 6.8 kHz. Spectralreferencing is with respect to an external sample of neattetramethylsilane, carried out by setting the high-frequency line fromadamantane to 38.5 ppm. Measurements are carried out in air and atambient probe temperature (~25° C.). The samples are analysedas-received. Particle Size PSD was determined using a Sympatec laserdistribution (PSD) diffraction HELOS/BF particle size instrument Laserdiffraction fitted with RODOS/ASPIROS dry dispersion unit operating at2.5 Bar with a sled speed of 25 mm/s, a combination of R1 0.1/0.18 μm-35μm and R3 0.5/0.9 μm-175 μm lenses were used for the determination.Trigger conditions: 1 ms, 0.2%. XRPD Bruker D2 Phaser X-Ray PowderDiffraction patterns are collected on a Bruker AXS D2 diffractometerusing Cu K radiation (30 kV, 10 mA), θ-θ geometry, using a Lynxeyedetector form 5-42 2θ. The software used for data collection is DIFFRAC.SUITE and the data are analysed and presented using Diffrac Plus EVA v13.0.0.2. Data collection: Angular range: 5 to 42 °20; Step size: 0.012°20; Collection time: 0.15 seconds per step. Sample Preparation: Samplesrun under ambient conditions are prepared as flat plate specimens usingpowder as received without grinding. Approximately 1-2 mg of the sampleis lightly pressed on a silicon wafer to obtain a flat surface.

Synthetic Preparation of the Salt of the Invention Example 1.Preparation of Compound 1 1.1. Route 1 1.1.1.4-[4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzyl]-thiomorpholine-1,1-dioxide

2-(4-Bromomethyl-phenyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (1 eq)and DIPEA (2 eq) are dissolved in DCM/MeOH (5:1 v:v) under N₂ andthiomorpholine 1,1-dioxide (2 eq) is added portionwise. The resultingsolution is stirred at room temperature for 16 h. After this time, thereaction is complete. The solvent is evaporated. The compound isextracted with EtOAc and water, washed with brine and dried overanhydrous MgSO₄. Organic layers are filtered and evaporated. The finalcompound is isolated without further purification.

1.1.2. Cyclopropanecarboxylic acid(5-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-amide

1.1.2.1. Step i): 1-(6-Bromo-pyridin-2-yl)-3-carboethoxy-thiourea

To a solution of 2-amino-6-bromopyridine (1) (253.8 g, 1.467 mol) in DCM(2.5 L) cooled to 5° C. is added ethoxycarbonyl isothiocyanate (173.0mL, 1.467 mol) dropwise over 15 min. The reaction mixture is thenallowed to warm to room temp. (20° C.) and stirred for 16 h. Evaporationin vacuo gives a solid which may be collected by filtration, thoroughlywashed with petrol (3×600 mL) and air-dried to afford the desiredproduct. The thiourea may be used as such for the next step without anypurification. ¹H (400 MHz, CDCl₃) δ 12.03 (1H, br s), 8.81 (1H, d), 8.15(1H, br s), 7.60 (1H, t), 7.32 (1H, dd), 4.31 (2H, q), 1.35 (3H, t).

1.1.2.2. Step ii): 5-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine

To a suspension of hydroxylamine hydrochloride (101.8 g, 1.465 mol) inEtOH/MeOH (1:1, 900 mL) is added N,N-diisopropylethylamine (145.3 mL,0.879 mol) and the mixture is stirred at room temp. (20° C.) for 1 h.1-(6-Bromo-pyridin-2-yl)-3-carboethoxy-thiourea (2) (89.0 g, 0.293 mol)is then added and the mixture slowly heated to reflux (Note: bleachscrubber is required to quench H₂S evolved). After 3 h at reflux, themixture is allowed to cool and filtered to collect the precipitatedsolid. Further product is collected by evaporation in vacuo of thefiltrate, addition of H₂O (250 mL) and filtration. The combined solidsare washed successively with H₂O (250 mL), EtOH/MeOH (1:1, 250 mL) andEt₂O (250 mL) then dried in vacuo to afford the triazolopyridinederivative (3) as a solid. The compound may be used as such for the nextstep without any purification. ¹H (400 MHz, DMSO-d₆) δ 7.43-7.34 (2H, m,2× aromatic-H), 7.24 (1H, dd, J 6.8 and 1.8 Hz, aromatic-H), 6.30 (2H,br, NH₂); m/z 213/215 (1:1, M+H⁺, 100%).

1.1.2.3. Step iii): Cyclopropanecarboxylic acid(5-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-amide

To a solution of the 2-amino-triazolopyridine obtained in the previousstep (7.10 g, 33.3 mmol) in dry MeCN (150 mL) at 5° C. is added Et₃N(11.6 mL, 83.3 mmol) followed by cyclopropanecarbonyl chloride (83.3mmol). The reaction mixture is then allowed to warm to ambienttemperature and stirred until all starting material is consumed. Ifrequired, further Et₃N (4.64 mL, 33.3 mmol) and cyclopropanecarbonylchloride (33.3 mmol) is added to ensure complete reaction. Followingsolvent evaporation in vacuo the resultant residue is treated with 7 Nmethanolic ammonia solution (50 mL) and stirred at ambient temp. (for1-16 h) to hydrolyse any bis-acylated product. Product isolation is madeby removal of volatiles in vacuo followed by trituration with Et₂O (50mL). The solids are collected by filtration, washed with H₂O (2×50 mL),acetone (50 mL) and Et₂O (50 mL), then dried in vacuo to give thedesired compound.

1.1.3. Compound 1

4-[4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzyl]-thiomorpholine-1,1-dioxide (1.1 eq.) is addedto a solution of cyclopropanecarboxylic acid(5-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-amide in 1,4-dioxane/water(4:1). K₂CO₃ (2 eq.) and PdCl₂dppf (0.03 eq.) are added to the solution.The resulting mixture is then heated in an oil bath at 90° C. for 16 hunder N₂. Water is added and the solution is extracted with ethylacetate. The organic layers are dried over anhydrous MgSO₄ andevaporated in vacuo.

The final compound is obtained after purification by flashchromatography.

Alternatively, after completion of the reaction, a palladium scavengersuch as 1,2-bis(diphenylphosphino)ethane, is added, the reaction mixtureis allowed to cool down and a filtration is performed. The filter cakeis reslurried in a suitable solvent (e.g. acetone), the solid isseparated by filtration, washed with more acetone, and dried. Theresulting solid is resuspended in water, aqueous HCl is added, and afterstirring at room temperature, the resulting solution is filtered oncelite (Celpure P300). Aqueous NaOH is then added to the filtrate, andthe resulting suspension is stirred at room temperature, the solid isseparated by filtration, washed with water and dried by suction. Finallythe cake is re-solubilised in a mixture of THF/H₂O, treated with apalladium scavenger (e.g. SMOPEX 234) at 50° C., the suspension isfiltered, the organic solvents are removed by evaporation, and theresulting slurry is washed with water and methanol, dried and sieved, toobtain the desired compound as a free base.

1.2. Route 2 1.2.1. Step 1: cyclopropanecarboxylic acid[5-(4-hydroxymethyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-amide

4-(Hydroxymethyl)phenylboronic acid (1.1 eq.) is added to a solution ofcyclopropanecarboxylic acid(5-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-amide in 1,4-dioxane/water(4:1). K₂CO₃ (2 eq.) and PdCl₂dppf (0.03 eq.) are added to the solution.The resulting mixture is then heated in an oil bath at 90° C. for 16 hunder N₂. Water is added and the solution is extracted with ethylacetate. The organic layers are dried over anhydrous MgSO₄ andevaporated in vacuo. The resulting mixture is used without furtherpurification.

1.2.2. Step 2: Cyclopropanecarboxylic acid[5-(4-bromomethyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-amide

To a solution of cyclopropanecarboxylic acid[5-(4-hydroxymethyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-amide(1.0 eq) in chloroform is slowly added phosphorus tribromide (1.0 eq.).The reaction mixture is stirred at room temperature for 20 h, quenchedwith ice and water (20 mL) and extracted with dichloromethane. Theorganic layer is dried over anhydrous MgSO₄, filtered and concentratedto dryness. The resulting white residue is triturated indichloromethane/diethyl ether 2:1 to afford the desired product.

Step 3

Cyclopropanecarboxylic acid [5-(4-bromomethyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-amide (1 eq) and DIPEA (2 eq) are dissolvedin DCM/MeOH (5:1 v:v) under N₂ and thiomorpholine 1,1-dioxide (1.1 eq)is added dropwise. The resulting solution is stirred at room temperaturefor 16 h. After this time, the reaction is complete. The solvent isevaporated. The compound is dissolved in DCM, washed with water anddried over anhydrous MgSO₄. Organic layers are filtered and evaporated.The final compound is isolated by column chromatography using EtOAc toafford the desired product.

Example 2. Preparation of the Salts of Compound 1 2.1. Protocol 1

Solvent is added to samples of Compound 1 (approximately 5 mg) inrelative volume portions of 10, 15 and 20 volumes, and after eachaddition the samples are agitated at 50° C. in a shaker for 20 min toencourage dissolution, before cooling to room temperature for the nextaddition of solvent. The solvent is selected from isopropanol, ethanol,methanol, isopropyl acetate, THF, TBME, acetone, methyl ethyl ketone,DCM, and MeCN.

Under these conditions, almost full dissolution of Compound 1 onlyoccurs in DCM.

After addition of 20 relative volumes of solvent, HCl (1 M solution inTHF) (12 μL˜1 eq.) is added to each sample and observations are made.The samples are stored in a shaker on a heat cool cycle (50° C./roomtemperature, 4 h at each temperature) for 16 h. The resultant solids areisolated by vacuum filtration, dried under suction and analysed by XRPD.

HCl salt formation is only observed in ethanol, methanol, THF, acetone,methyl ethyl ketone, and MeCN.

2.2. Protocol 2

Compound 1 (˜20 mg) is treated with 400 μL (20 volumes) of solvent (THF,acetone) and treated with aqueous acid solutions at room temperature(see Table III below). The experiments are placed in a maturationchamber which is cycled between ambient temperature and 50° C. with fourh spent under each condition. After three days solids are isolated byfiltration and any solutions are allowed to evaporate. Any furthersolids formed are isolated, or remaining oils are treated with EtOAc andplaced back in the maturation chamber for four days. Any further solidsare isolated by filtration.

The solids are analysed by XRPD.

When subjected to this protocol, salts are only formed in sulfuric acid,pTSA, 1,2-ethane disulfonic acid, 2-hydroxyethanesulfonic acid,naphthalene 2-sulfonic acid, and maleic acid.

TABLE III Acids used in Protocol 2 Acid Solvent Eq. Acid 1-5-Naphthalenedisulfonic acid Water 2.1 sulfuric acid Water 2.1 1-2-Ethane disulfonicacid Water 2.1 p-Toluene sulfonic acid Water 2.1 Methane sulfonic acidWater 2. 1 1-5-Naphthalene disulfonic acid Water 1.1 sulfuric acid Water1.1 1-2-Ethane disulfonic acid Water 1.1 p-Toluene sulfonic acid Water1.1 Methane sulfonic acid Water 1.1 Naphthalene-2-sulfonic acid Water1.1 Benzene sulfonic acid Water 1.1 Oxalic acid Water 1.1 2-Hydroxyethanesulfonic acid Water 1.1 Maleic acid Water 1.1 Phosphoric acidWater 1.1 Ethane sulfonic acid Water 1.1 Malonic acid Water 1.12-5-Dihydroxybenzoic acid THF 1.1 L-Tartaric acid Water 1.1

2.3. Protocol 3

To a suspension of Compound 1 (˜25 mg) and solvent (MeOH, THF, Acetone,or DCM) (20 relative volumes) at approximately 40° C. is added 1.05 or2.1 eq. of the acid (HCl, HBr, H₂SO₄, Ph-SO₃H, oxalic acid, maleic acid,orpTSA.H₂O), and visual observations are made. The samples are stored ina shaker at 26° C. for 14 h. An aliquot is taken and the solid isisolated by vacuum filtration, dried under suction and analysed by XRPD.The mixtures are then stored in a shaker at 26° C. for a further 31 h.The remaining solid from those samples exhibiting crystalline XRPDpatterns different to the free base are isolated by vacuum filtrationand further analysis is done (DSC, NMR, Stability).

The remainder of each sample is stored in a shaker on a heat/cool cycle(50° C./room temperature, 4 h at each temperature) for 136 h. Again, analiquot of each sample is taken and the solid is isolated by vacuumfiltration, dried under suction and analysed by XRPD. The remainingsolid from those samples exhibiting crystalline XRPD patterns differentto the free base is isolated by vacuum filtration and further analysisis undertaken (DSC, NMR, Stability).

Samples that remain in solution are allowed to slowly evaporate underambient conditions until the solvent has evaporated, and any solidformed is isolated and analysed by XRPD.

When subjected to this protocol, salts are only formed in HBr, HCl,sulfuric acid, pTSA, and maleic acid.

2.4. Protocol 4 2.4.1. HCl

HCl (1 M solution in THF) (655 μL, 0.65 mmol, 1.1 eq.) is added to astirred suspension of Compound 1 (252.6 mg, 0.59 mmol, 1 eq.) andmethanol (5.05 mL, 20 vols) at 50° C. The mixture is cooled to 25° C. at1° C./min and stirred at 25° C. for 22 h.

The solid is isolated by vacuum filtration and dried under suction.

The XRPD analysis confirmed the formation of a stable non-hygroscopicsalt, containing 4-5% water, having an aqueous solubility measured at1.9 mg/mL.

2.4.2. Maleic Acid

Compound 1 (246.4 mg, 0.58 mmol, 1 eq.) is suspended in 5% water inacetone (4.95 mL, 20 vols) at 25° C. and the sample is warmed to 50° C.Maleic acid (1 M solution in THF) (1.2 mL, 1.22 mmol, 2.1 eq.) is addedto the stirred suspension at 50° C. The mixture is cooled to 25° C. at1° C./min and stirred at 25° C. for 22 h.

The solid was isolated by vacuum filtration and dried under suction.

The XRPD analysis confirmed the formation of the formation of a stablenon-hygroscopic salt, having an aqueous solubility measured at 0.4mg/mL.

2.4.3. pTSA

pTSA (1 M solution in EtOH) (1.3 mL, 1.3 mmol, 2.2 eq.) is added to astirred suspension of Compound 1 (250.7 mg, 0.59 mmol, 1 eq.) and THF (5mL, 20 vols) at 50° C. The mixture is cooled to 25° C. at 1° C./min andstirred at 25° C. for 22 h.

The solid is isolated by vacuum filtration and dried under suction.

The XRPD analysis confirmed the formation of a salt, which appeared tobe unstable.

2.5. Complementary Salt Analysis

The solids recovered from this protocol are also subjected to purity,salt equivalent (IC), NMR (residual solvent), DSC, TGA (solvates), andstability evaluation (1 week at 40° C./75% Relative humidity).

2.5.1. pKa Determination

Data are collected on a Sirius G1pKa instrument with a D-PAS attachment.Measurements are made at 25° C. in aqueous solution by UV and inmethanol water mixtures by potentiometry. The titration media wasionic-strength adjusted with 0.15 M KCl (aq). The values found in themethanol water mixtures are corrected to 0% co-solvent via aYasuda-Shedlovsky extrapolation.

The data are refined using Refinement Pro software.

Following this procedure, the following values are obtained: 1.58±0.01,3.68±0.02, and 12.02±0.01.

2.5.2. pH Determination

The pH measurements are performed on the test material as a slurry.Typically 1 mL of demineralized water is added to about 300 mg of thetested material and the suspension is then vortexed. If insufficientliquid is available or viscosity is too high for measurement of pH usinga pH electrode, the amount of water is increased until a measurement ispossible in increments of 1 ml. The pH electrode was calibrated using athree point calibration.

Finally, pH is recorded at start (=after 2 min to allow a relativelystable measurement of the pH).

2.5.3. Solubility Study 2.5.3.1. Protocol

The solubility of a compound is determined in different solvents byequilibrating an excess of product for at least 24 h at 20° C. on arotary shaker. The supersaturated solution is then filtered and theconcentration of product in the filtrate is determined usingUV-spectrometry.

In water, the influence of the pH is adjusted to pH 2, pH 7, and pH 9.HCl and NaOH are used to adjust the pH.

2.5.3.2. Results

For example, when subjected to this protocol, the solubility of the freebase and the corresponding HCl salt measurement are reported in Table IVbelow. Unexpectedly, going from Compound 1 to Compound 1.HCl.3H₂O doesnot systematically result in an improved solubility.

TABLE IV Solubility of Compound 1 as a free base and the correspondingHCl salt in various solvents Compound 1 Solubility Compound1•HCl•3H₂OSolvent (mg/mL) Solubility (mg/mL) Acetone 0.420 0.415 Acetonitrile0.605 7.36 Acetonitrile/water (1/1) 1.57 25.6 Acetonitrile/water (4/1)3.11 7.81 Dichloromethane 5.66 0.388 Ethanol 0.151 0.764 0.005M HCl0.252 3.10  0.01M HCl 0.288 2.34 0.01M HCl + 2.0% Tweet 80 0.313 2.79HP-β-CD 40% pH2 0.577 7.88 HP-β-CD 40% pH3 0.550 9.33 HP-β-CD 40% pH40.520 2.79 Methanol 0.432 0.743 PEG400 1.53 36.6 Propanol 0.134 0.663Propylene glycol 0.464 11.7 t-butyl methyl ether <0.00239 0.0263Tetrahydrofuran 0.740 0.208 Water (purified) 0.00384 3.17 Water pH20.291 8.05 Water pH7 0.00432 2.26 Water pH9 0.00429 2.68 Water/ethanol1/1 0.327 8.68

2.5.4. Conclusions

As demonstrated above, the solubility of Compound 1 varies greatlydepending on the solvent used, and salt formation does not inevitablyoccurs with every acid, illustrating the difficulty in selecting asatisfactory combination of solvent and acid, which is specific toCompound 1.

Example 3. Polymorphism Study 3.1. Compound 1 3.1.1. Amorphous Compound1 Formation

Compound 1 (38 mg) is heated in a DSC instrument under nitrogenaccording to the following procedure: 1) Heated from 30° C. to 250° C.at 10° C./min; 2) Held isothermal for 1 min; 3) Cooled from 250° C. to30° C. at 100° C./min; and 4) Held isothermal for 4 min.

The resulting solid is confirmed to be amorphous by XRPD analysis.

3.1.2. Polymorphism Study

Amorphous compound was subjected to forty different solvents (ethylformate, TBME, acetone, methyl acetate, methanol, tetrahydrofuran,diisopropyl ether, ethyl acetate, ethanol, methylethyl ketone,acetonitrile, t-BuOH, 2-Propanol, 1-2-Dimethoxyethane, IsopropylAcetate, 1-Propanol, 2-Butanol, nitromethane, 1-4-Dioxane, propylacetate, 2-Pentanone, 2-Methyl-1-Propanol, Toluene, Isobutyl Acetate,Methylisobutyl Ketone, 1-Butanol, 2-Methoxyethanol, Butyl Acetate,Methylbutyl Ketone, 3-Methyl-1-Butanol, 2-Ethoxyethanol, 1-Pentanol,Anisole, Benzonitrile, nitrobenzene, IPA+5% water, EtOH+5% water,MeCN+5% water, THF+5% water, and acetone+5% water) and thermally cycledbetween ambient and 50° C. with four h spent under each condition. Afterfive days the solids are collected by filtration and initially analysedby XRPD.

3.1.3. Compound 1 Polymorphism Results

When subjected to this protocol, the obtained solids were analysed byXRPD, and the following most stable patterns are reported in the TableV, Table VI, and Table VII below.

TABLE V Compound 1 Pattern 1 XRPD pattern (Error! Reference source notfound.) Angle (2θ°) Intensity (%) 16.8 100.0 9.4 30.4 9.6 14.0 13.1 26.214.3 12.2 17.3 12.4 18.4 9.5 18.7 13.0 19.0 43.8 20.6 9.1 20.8 15.3 21.320.5 23.8 18.0 24.6 14.3 25.2 5.8 29.7 8.7

TABLE VI Compound 1 Pattern 3 XRPD pattern (Error! Reference source notfound.) Angle (2θ°) Intensity (%) 8.6 52 9.7 24.6 10.6 35.5 13.0 39.415.3 61.7 16.9 40 17.3 45.6 17.7 50.3 17.9 14.1 18.3 10.9 18.9 100 19.321.4 19.6 12.6 19.8 28.1 20.0 28.7 20.3 13.9 20.8 52.2 23.0 37 23.5 15.423.9 50.1 24.4 23.8 25.0 14.1 29.1 15 33.5 32.3 34.9 23.6

TABLE VII Compound 1 Pattern 4 XRPD pattern (Error! Reference source notfound.) Angle (2θ°) Intensity (%) Angle (2θ°) Intensity (%) 7.2 100 15.313.3 8.2 56.9 16.4 26.4 10.9 85 17.4 61.3 14.4 23.5 18.4 75.7 18.5 89.622.7 24.7 18.8 69.6 25.4 55.2 19.2 32.3 27.5 47.6 19.9 58.6 28.2 18.620.2 46.1 30.7 13.9 20.5 42.9 32.5 16.3 21.8 18.4

3.2. Polymorphism of HCl Salts of Compound 1 and Solvates Thereof 3.2.1.Amorphous HCl Salts of Compound 1 Formation

Compound 1 (25.47 mg) is mixed with water (70 mL, 2750 relative volumes)and stirred at room temperature for 30 min. The mixture is filteredthrough a 0.45 mm PVDF membrane syringe filter and frozen in a −78° C.bath. The solvent is removed using a freeze drier to yield amorphousCompound 1 mono-HCl salt (confirmed to by XRPD, glass transition at 149°C. (modulated DSC analysis))

3.2.2. Study Protocols

Volumes of 25 different solvents (Acetone, anisole, butanol, butylacetate, TBME, DMSO, ethanol, ethyl acetate, heptane, isopropyl acetate,MEK, isopropyl acetate, MeCN, cyclohexane, DCM, dioxane, methanol,nitromethane, THF, methyl THF, toluene, water, 10% aqueous acetone, 10%aqueous THF, and 10% methanol), are added to amorphous mono-HCl salt ofCompound 1, said volumes ranging from 125 μL (5 rel vol) to 1 mL (40 relvol), or until a mobile suspension or almost complete dissolution isobserved. The sample is stored in a shaker on a heat/cool cycle (50°C./room temperature, 4 h) for 23 h.

After 23 h, where solid has formed, an aliquot is taken and the solid isisolated by vacuum filtration, whereas the remaining suspension is keptin a shaker on a heat/cool cycle (50° C./room temperature, 4 h). Thesolid from the aliquot is dried under suction for 30 min and analysed byXRPD, then dried further in a vacuum oven at 30° C. and 5 mbar for 34 hand re-analysed by XRPD. Finally, the dried sample is stored at 40°C./75% RH for 72 h and re-analysed by XRPD.

After 47 h, where solid has formed in the suspension, an aliquot istaken and the solid is isolated by vacuum filtration, whereas theremaining suspension is kept in a shaker on a heat/cool cycle (50°C./room temperature, 4 h). The solid from the aliquot is stored underambient conditions for 72 h and 14 d with analysis by XRPD at each timepoint. The solid, which has been stored under ambient conditions for 14d, is stored at 40° C./75% RH for 43 h and re-analysed by XRPD.

After 143 h, where solid has formed, an aliquot is taken from theremaining suspension stored in a shaker on a heat/cool cycle (50°C./room temperature, 4 h) and the solid is isolated by vacuumfiltration, dried under suction for 2 h, then analysed by XRPD. Theisolated solid is dried in a vacuum oven at 30° C. and 5 mbar for 20 hand re-analysed by XRPD. The dried sample is stored at 40° C./75% RH for114 h and re-analysed by XRPD.

3.2.3. Results

Further to this study, two stable forms are identified.

3.2.3.1. Compound 1.HCl

HCl (3.6 M solution in dioxane) (2.5 mL, 9.05 mmol 1.1 eq.) is addedportion-wise to a stirred suspension of amorphous Compound 1 (3.499 g,8.22 mmol, 1 eq.) and methanol (70 mL, 20 relative volumes) at 50° C.over a period of 2 min. The mixture is cooled to 20° C. at 0.1° C./minand stirred at 20° C. for a further 10 h. The mixture is cooled to 15°C. at 0.5° C./min and stirred for 30 min. The resultant solid isisolated by vacuum filtration, washed with methanol (2×3.5 mL, 1×7 mL)and dried under suction to yield Compound 1.HCl.

3.2.3.2. Compound 1.HCl.3H₂O

HCl (1 M solution in THF) (250 μL, 0.25 mmol, 1.05 eq.) is added to asuspension of amorphous Compound 1 (100.37 mg, 0.24 mmol, 1 eq.) and DCM(2 mL, 20 relative volumes) at 40° C. The mixture is stored in a shakeron a heat/cool cycle (40° C./room temperature, 4 h) for 70 h. The solidis isolated by vacuum filtration and dried under suction to yieldCompound 1.HCl.3H₂O.

3.2.3.3. Analysis

The X-Ray diffraction patterns are disclosed in Table VIII and Table IXbelow.

TABLE VIII Compound 1. HCl XRPD pattern (Error! Reference source notfound.) Angle (2θ°) Intensity (%) Angle (2θ°) Intensity (%) 7.4 100.020.7 43.4 8.9 15.6 21.1 11.9 12.4 21.3 22.8 14.6 14.8 61.1 24.9 12.315.1 31.7 26.0 12.3 16.9 15.9 28.6 14.2 17.6 27.7 29.8 27.3 19.4 11.432.6 10.2

TABLE IX Compound 1. HCl•3H₂O XRPD pattern (Error! Reference source notfound.) Angle (2θ°) Intensity (%) Angle (2θ°) Intensity (%) 7.3 61.413.7 16.1 8.4 35.3 14.5 14.0 8.8 62.8 16.3 31.0 10.7 26.3 16.7 100.012.0 22.5 17.6 11.3 12.2 18.1 19.3 20.8 13.2 23.6 20.2 87.5 20.6 16.425.9 33.1 21.0 18.0 26.4 11.7 21.4 52.0 27.2 13.0 21.8 58.8 27.7 16.622.8 45.0 28.3 10.8 23.4 57.5 28.6 19.3 23.9 10.6 28.9 17.2 24.5 10.829.2 20.2 25.2 21.7 29.6 47.6 25.7 35.3 32.7 26.1

3.2.3.4. Compound 1.HCl.3H₂O Single Crystal X-Ray Diffraction (Error!Reference Source not Found.)

Compound 1.HCl.3H₂O is recrystallized from acetone:water (1:1). Theresults are disclosed in Table X below.

TABLE X Single Crystal structure of Compound 1. HCl•3H₂O Molecularformula C₂₁H₂₄N₅O₃S•Cl•₃(H₂O) Molecular weight 516.02 Crystal systemMonoclinic Space group P2_(1/n) a 13.1388 (4) Å α 102.089(2)° b  8.9437(3) Å β c 21.6376 (9) A γ V 2486.24(15) Å³ Z 4 Dc 1.379 mg/m³ μ 0.284mm⁻¹ Source Mo-K α, 0.71073 Å F(000) 1088 T 120(2) K Crystal colourlessprism, 0.39 × 0.16 × 0.12 mm θ range for data collection 2.982-27.483°Completeness 99.3% Reflections 21028 Unique reflections 5649 R_(int)0.0307

Refinement method is based on Full-matrix least-squares on F².R[F²>2σ(F²)]=0.0377 and wR(F²)=0.0837. Goodness of fit (S)=1.109. Therefinement method used 5649 reflections, 339 parameters and 0restraints. All hydrogen positions were identified using the differencemap and those attached to C atoms & N atoms were then placed incalculated positions and refined using a riding model. Those hydrogen'sattached to the water oxygen's and nitrogen were freely refined. Thefinal Δ_(ρmax)=0.314 e Å⁻³ and Δ_(ρmin)=−0.368 e Å⁻³.

The crystal structure of Compound 1.HCl.3H₂O (Error! Reference sourcenot found.) shows the unexpected inclusion of the water molecules in thecrystal lattice which may provide further stabilisation of the system.

Example 4. Large Scale Compound 1.HCl.3H₂O Formation 4.1. Protocol 1

To Compound 1 (44 kg, 1.0 eq) under inert atmosphere, is added water (15rel vol, 1000 L), and the mixture is stirred at 50° C. 3.5 eq. aq HCl (5rel vol) is added over 10-15 min, at a maximum temperature of 55° C.Upon completion of the addition, the stirring is continued at 50° C. for15 min, and the reaction is then cooled to 15° C. and stirred at thattemperature for at least 12 h but no more than 24 h.

The resulting solid is separated by filtration, and the cake is washedwith water (2.0 rel vol), and the cake is dried under nitrogen for atleast 4 h to afford the desired product.

4.2. Protocol 2

To Compound 1 (45 g, 106 mmol, 1 eq.) under inert atmosphere is addedDCM (675 mL) and methanol (225 mL). The resulting suspension is heatedto 35° C. under stirring, and trimercaptotriazine trisodium salt 15% inwater (22.5 g, 14 mmol, 0.13 eq) is added, and the resulting solution isstirred for 5 h, after which the solution is filtered on 0.45 μm paperunder nitrogen pressure.

To the filtrate is added water (50 mL), and the resulting biphasicmixture is stirred at 35° C. for 15 min, after which period the phasesare separated, and the organic layer is allowed to cool down to 20° C.,and washed twice more with 50 mL water.

The organic layer is cooled down to 15-20° C., then HCl 10% in methanol(42.4 g, 116 mmol, 1.10 eq.) is added over 30 min, causing theprecipitation of a solid. The suspension is further stirred at 20° C.for 3 h, then the precipitate is isolated by filtration, the cake iswashed with methanol (2×50 mL) to afford the desired compound, which isdried under vacuum at 45° C. for 3 h. The cake is then resuspended inwater (220 mL) and stirred for 6 h at 50° C., and then cooled to 15-20°C. The resulting solid is separated by filtration and the cake is washedwith water (2×30 mL), and dried at 45° C. for 3 h to afford the desiredproduct.

4.3. Protocol 3 4.3.1. Step 1: Compound 1.HCl.MeOH

To Compound 1 (100 g, 235 mmol, 1 eq.) suspended in DCM (1.5 L), isadded MeOH (0.5 L), and the resulting solution is heated to 35° C.Trimercaptotriazine trisodium 85% (8.7 g, 3 mmol, 0.13 eq.) in water (42mL) is added and the resulting mixture is stirred at 35° C. for at least5 h. The solution is then filtered on a 0.45 μm paper filter undernitrogen pressure.

To the resulting solution is added water (150 g), stirred at 35° C. for15 to 30 min, and the biphasic mixture is separated. The organic layeris washed again twice with water (2×150 g).

Finally, a solution of HCl in MeOH (10% w/w) (141 g) is added, and thesuspension is stirred at 20° C. for 3 h, and the resulting solid isseparated by filtration, the cake is washed with MeOH (2×118 g), driedunder vacuum for 3 h at 45° C., to afford Compound 1.HCl.MeOH which isanalysed by XRPD. (Table XI).

4.3.2. Step 2: Compound 1.HCl.3H₂O

To formic acid (200 g, 1.6 eq) in water (36 g, 0.4 eq.) is addedCompound 1.HCl.MeOH (100 g, 1 eq.) obtained in Step 1 above. Theresulting mixture is heated to 55° C. under stirring, and the solutionis filtered through a 0.45 μm filter cartridge. Formic acid 85% aq (200g) is added, and the mixture is cooled to 28-32° C. under gentlestirring.

Water (100 g) is then added, followed with Compound 1.HCl.3H₂O (1 g)causing the precipitation of Compound 1.HCl.1.5HCO₂H, which is analysedby XRPD (Table XII).

Under stirring at 28-32° C., water (2 L) is added portionwise in 8portions of 100 mL, 1 portion of 200 mL, and 2 portions of 500 mL.

The resulting suspension is then filtered, the cake is washed with water(2×100 mL) and dried at 30-35° C. to yield Compound 1.HCl.3H₂O, which isanalysed by XRPD and DSC. (Error! Reference source not found. and Error!Reference source not found.).

TABLE XI Compound 1 HCl•MeOH XRPD pattern (Error! Reference source notfound.) Angle (2θ°) Intensity (%) Angle (2θ°) Intensity (%) 7.1 100 18.94.3 14.4 37.9 23.4 4.8 16.6 8.9 24.8 5.1 17.3 5.1 29.0 10.4

TABLE XII Compound 1. HCl•HCOOH XRPD pattern (Error Reference source notfound.) Angle (2θ°) Intensity (%) Angle (2θ°) Intensity (%) 7.1 100 20.813.1 14.4 76.4 23.4 14.1 14.8 17.1 24.5 16.1 16.4 25.1 24.9 13.4 17.442.8 29.0 39.8 18.6 20.6

Example 5. Compound 1.HCl.3H₂O Stability Study 5.1. AcceleratedStability Study 5.1.1. Protocol

Samples of Compound 1.HCl.3H₂O are stored under conditions to evaluatechemical stability and physical stability as described in Table XIII.

TABLE XIII Stability conditions Chemical stability conditions Physicalstability conditions 25° C./60% RH/Open recipient RT/<5% RH/Openrecipient 40° C./75% RH/Open recipient RT/56% RH/Open recipient 50°C./Closed recipient RT/75% RH/Open recipient

Samples are taken at TO, then every month up to 3 months, and analysedby FT-IR, DSC, and XRPD.

5.2. Extended Stability Study 5.2.1. Protocol

Samples of test compounds are stored under conditions to evaluatechemical stability and physical stability as described in Table XIVbelow:

TABLE XIV Stability conditions Model Temp (° C.)/RH (%)/recipient Coldconditions  5° C./_/Closed recipient Long term storage 25° C./60%RH/Open recipient Intermediate conditions 30° C./65% RH/Open recipientAccelerated conditions 40° C./75% RH/Open recipient

Samples are taken at TO, then every month up to 12 months, then at 18,24 and 36 months; and each aliquots is then analysed by HPLC,Karl-Fisher titration, FT-IR, DSC, and XRPD.

5.3. GVS Analysis Compound 1.HCl.3H₂O

GVS analysis is carried out in order to assess the stability of Compound1.HCl.3H₂O and is presented on Error! Reference source not found. Theevolution of the material is followed via XPRD at various time points.

No change in form is observed at elevated RH. Compound 1.HCl.3H₂Odehydrates to Compound 1.HCl anhydrous upon heating above 60° C. or atless than 10% RH. Rehydration to Compound 1.HCl.3H₂O occurs upon coolingto ambient temperature or by exposure to 20% or greater relativehumidity.

5.3.1. Conclusions

Surprisingly, Compound 1.HCl.3H₂O can be dehydrated to Compound 1.HCl,but converts back to the more stable trihydrate form Compound 1.HCl.3H₂Ounder normal conditions. This could not have been predicted by theskilled person.

BIOLOGICAL EXAMPLES Example 6. In Vitro Assays 6.1. JAK1 InhibitionAssay

Recombinant human JAK1 catalytic domain (amino acids 850-1154; catalognumber 08-144) is purchased from Carna Biosciences. 10 ng of JAK1 isincubated with 12.5 μg polyGT substrate (Sigma catalog number P0275) inkinase reaction buffer (15 mM Tris-HCl pH 7.5, 1 mM DTT, 0.01% Tween-20,10 mM MgCl₂, 2 μM non-radioactive ATP, 0.25 μCi ³³P-gamma-ATP (GEHealthcare, catalog number AH9968) final concentrations) with or without5 μL containing test compound or vehicle (DMSO, 1% final concentration),in a total volume of 25 μL, in a polypropylene 96-well plate (Greiner,V-bottom). After 45 min at 30° C., reactions are stopped by adding of 25μL/well of 150 mM phosphoric acid. All of the terminated kinase reactionis transferred to prewashed (75 mM phosphoric acid) 96 well filterplates (Perkin Elmer catalog number 6005177) using a cell harvester(Perkin Elmer). Plates are washed 6 times with 300 μL per well of a 75mM phosphoric acid solution and the bottom of the plates is sealed. 40μL/well of Microscint-20 is added, the top of the plates is sealed andreadout is performed using the Topcount (Perkin Elmer). Kinase activityis calculated by subtracting counts per minute (cpm) obtained in thepresence of a positive control inhibitor (10 μM staurosporine) from cpmobtained in the presence of vehicle. The ability of a test compound toinhibit this activity is determined as:

Percentage inhibition=((cpm determined for sample with test compoundpresent−cpm determined for sample with positive control inhibitor)divided by (cpm determined in the presence of vehicle−cpm determined forsample with positive control inhibitor))*100.

Dose dilution series are prepared for the compounds enabling the testingof dose-response effects in the JAK1 assay and the calculation of theIC₅₀ for each compound. Each compound is routinely tested atconcentration of 20 μM followed by a 1/3 serial dilution, 8 points (20μM-6.67 μM-2.22 μM-740 nM-247 nM-82 nM-27 nM-9 nM) in a finalconcentration of 1% DMSO. When potency of compound series increased,more dilutions are prepared and/or the top concentration is lowered(e.g. 5 μM, 1 μM).

The following compounds have been tested for their activity against JAK1and the IC₅₀ values, as determined using the assays described herein,are given below in Table XV.

TABLE XV JAK1 IC₅₀ Values of Compounds Cpd # JAK1 IC₅₀ (nM) 1 47.07,55.66, 50.1, 48.29

6.2. JAK1 Ki Determination Assay

For the determination of Ki, different amounts of compound are mixedwith the enzyme and the enzymatic reaction is followed as a function ofATP concentration. The Ki is determined by means of double reciprocalplotting of Km vs compound concentration (Lineweaver-Burk plot). 1 ng ofJAK1 (Invitrogen, PV4774) is used in the assay. The substrate is 50 nMUlight-JAK-1 (Tyr1023) Peptide (Perkin Elmer, TRF0121) The reaction isperformed in 25 mM MOPS pH 6.8, 0.01%, 2 mM DTT, 5 mM MgCl₂ Brij-35 withvarying concentrations of ATP and compound. Phosphorylated substrate ismeasured using an Eu-labeled anti-phosphotyrosine antibody PT66 (PerkinElmer, AD0068). Readout is performed on the envision (Perkin Elmer) withexcitation at 320 nm and emission followed at 615 nm and 665 nm.

For example, when Compound 1 is tested in this assay, a Ki value of 39nM is measured.

6.3. JAK2 Inhibition Assay

Recombinant human JAK2 catalytic domain (amino acids 808-1132; catalognumber PV4210) is purchased from Invitrogen. 0.025 mU of JAK2 isincubated with 2.5 μg polyGT substrate (Sigma catalog number P0275) inkinase reaction buffer (5 mM MOPS pH 7.5, 9 mM MgAc, 0.3 mM EDTA, 0.06%Brij and 0.6 mM DTT, 1 μM non-radioactive ATP, 0.25 μCi ³³P-gamma-ATP(GE Healthcare, catalog number AH9968) final concentrations) with orwithout 5 μL containing test compound or vehicle (DMSO, 1% finalconcentration), in a total volume of 25 μL, in a polypropylene 96-wellplate (Greiner, V-bottom). After 90 min at 30° C., reactions are stoppedby adding of 25 μL/well of 150 mM phosphoric acid. All of the terminatedkinase reaction is transferred to prewashed (75 mM phosphoric acid) 96well filter plates (Perkin Elmer catalog number 6005177) using a cellharvester (Perkin Elmer). Plates are washed 6 times with 300 μL per wellof a 75 mM phosphoric acid solution and the bottom of the plates issealed. 40 μL/well of Microscint-20 is added, the top of the plates issealed and readout is performed using the Topcount (Perkin Elmer).Kinase activity is calculated by subtracting counts per minute (cpm)obtained in the presence of a positive control inhibitor (10 μMstaurosporine) from cpm obtained in the presence of vehicle. The abilityof a test compound to inhibit this activity is determined as:

Percentage inhibition=((cpm determined for sample with test compoundpresent−cpm determined for sample with positive control inhibitor)divided by (cpm determined in the presence of vehicle−cpm determined forsample with positive control inhibitor))*100.

Dose dilution series are prepared for the compounds enabling the testingof dose-response effects in the JAK2 assay and the calculation of theIC₅₀ for each compound. Each compound is routinely tested atconcentration of 20 μM followed by a 1/3 serial dilution, 8 points (20μM-6.67 μM-2.22 μM-740 nM-247 nM-82 nM-27 nM-9 nM) in a finalconcentration of 1% DMSO. When potency of compound series increased,more dilutions are prepared and/or the top concentration is lowered(e.g. 5 μM, 1 μM).

The following compounds have been tested for their activity against JAK2and the IC₅₀ values, as determined using the assays described herein,are given below in Table XVI.

TABLE XVI JAK2 IC₅₀ Values of Compounds Cpd # JAK2 IC₅₀ (nM) 1 31.37,41.16, 55.49, 167.34

6.4. JAK2 Kd Determination Assay

JAK2 (Invitrogen, PV4210) is used at a final concentration of 5 nM. Thebinding experiment is performed in 50 mM Hepes pH 7.5, 0.01% Brij-35, 10mM MgCl₂, 1 mM EGTA using 25 nM kinase tracer 236 (invitrogen, PV5592)and 2 nM Eu-anti-GST (Invitrogen, PV5594) with varying compoundconcentrations. Detection of tracer is performed according to themanufacturer's procedure.

For example, when Compound 1 is tested in this assay, a Kd value of 205nM is measured.

6.5. JAK3 Inhibition Assay

Recombinant human JAK3 catalytic domain (amino acids 781-1124; catalognumber PV3855) is purchased from Invitrogen. 0.025 mU of JAK3 isincubated with 2.5 μg polyGT substrate (Sigma catalog number P0275) inkinase reaction buffer (25 mM Tris pH 7.5, 0.5 mM EGTA, 0.5 mM Na₃VO₄, 5mM b-glycerolphosphate, 0.01% Triton X-100, 1 μM non-radioactive ATP,0.25 μCi 33P-gamma-ATP (GE Healthcare, catalog number AH9968) finalconcentrations) with or without 5 μL containing test compound or vehicle(DMSO, 1% final concentration), in a total volume of 25 μL, in apolypropylene 96-well plate (Greiner, V-bottom). After 105 min at 30°C., reactions are stopped by adding of 25 μL/well of 150 mM phosphoricacid. All of the terminated kinase reaction is transferred to prewashed(75 mM phosphoric acid) 96 well filter plates (Perkin Elmer catalognumber 6005177) using a cell harvester (Perkin Elmer). Plates are washed6 times with 300 μL per well of a 75 mM phosphoric acid solution and thebottom of the plates is sealed. 40 μL/well of Microscint-20 is added,the top of the plates is sealed and readout is performed using theTopcount (Perkin Elmer). Kinase activity is calculated by subtractingcounts per minute (cpm) obtained in the presence of a positive controlinhibitor (10 μM staurosporine) from cpm obtained in the presence ofvehicle. The ability of a test compound to inhibit this activity isdetermined as:

Percentage inhibition=((cpm determined for sample with test compoundpresent−cpm determined for sample with positive control inhibitor)divided by (cpm determined in the presence of vehicle−cpm determined forsample with positive control inhibitor))*100.

Dose dilution series are prepared for the compounds enabling the testingof dose-response effects in the JAK3 assay and the calculation of theIC50 for each compound. Each compound is routinely tested atconcentration of 20 μM followed by a 1/3 serial dilution, 8 points (20μM-6.67 μM-2.22 μM-740 nM-247 nM-82 nM-27 nM-9 nM) in a finalconcentration of 1% DMSO. When potency of compound series increased,more dilutions are prepared and/or the top concentration is lowered(e.g. 5 μM).

The following compounds have been tested for their activity against JAK3and the IC₅₀ values, as determined using the assays described herein,are given below in Table XVII.

TABLE XVII JAK3 IC₅₀ Values of Compounds Cpd # JAK3 IC₅₀ (nM) 1 149.35,187.3, 189.3, 194.7

6.6. JAK3 Ki Determination Assay

For the determination of Ki, different amounts of compound are mixedwith the enzyme and the enzymatic reaction is followed as a function ofATP concentration. The Ki is determined by means of double reciprocalplotting of Km vs compound concentration (Lineweaver-Burk plot). JAK3(Carna Biosciences, 09CBS-0625B) is used at a final concentration of 10ng/mL. The substrate is Poly(Glu,Tyr)sodium salt (4:1), MW 20 000-50 000(Sigma, P0275) The reaction is performed in 25 mM Tris pH 7.5, 0.01%Triton X-100, 0.5 mM EGTA, 2.5 mM DTT, 0.5 mM Na3VO4, 5 mMb-glycerolphosphate, 10 mM MgCl2 with varying concentrations of ATP andcompound and stopped by addition of 150 mM phosphoric acid. Measurementof incorporated phosphate into the substrate polyGT is done by loadingthe samples on a filter plate (using a harvester, Perkin Elmer) andsubsequent washing. Incorporated 33P in polyGT is measured in a Topcountscintillation counter after addition of scintillation liquid to thefilter plates (Perkin Elmer).

For example, when Compound 1 is tested in this assay, a Ki value of 353nM is measured.

6.7. TYK2 Inhibition Assay

Recombinant human TYK2 catalytic domain (amino acids 871-1187; catalognumber 08-147) is purchased from Carna biosciences. 5 ng of TYK2 isincubated with 12.5 μg polyGT substrate (Sigma catalog number P0275) inkinase reaction buffer (25 mM Hepes pH 7.5, 100 mM NaCl, 0.2 mM Na₃VO₄,0.1% NP-40, 0.1 μM non-radioactive ATP, 0.125 μCi ³³P-gamma-ATP (GEHealthcare, catalog number AH9968) final concentrations) with or without5 μL containing test compound or vehicle (DMSO, 1% final concentration),in a total volume of 25 μL, in a polypropylene 96-well plate (Greiner,V-bottom). After 90 min at 30° C., reactions are stopped by adding 25μL/well of 150 mM phosphoric acid. All of the terminated kinase reactionis transferred to prewashed (75 mM phosphoric acid) 96 well filterplates (Perkin Elmer catalog number 6005177) using a cell harvester(Perkin Elmer). Plates are washed 6 times with 300 μL per well of a 75mM phosphoric acid solution and the bottom of the plates is sealed. 40μL/well of Microscint-20 is added, the top of the plates is sealed andreadout is performed using the Topcount (Perkin Elmer). Kinase activityis calculated by subtracting counts per minute (cpm) obtained in thepresence of a positive control inhibitor (10 μM staurosporine) from cpmobtained in the presence of vehicle. The ability of a test compound toinhibit this activity is determined as:

Percentage inhibition=((cpm determined for sample with test compoundpresent−cpm determined for sample with positive control inhibitor)divided by (cpm determined in the presence of vehicle−cpm determined forsample with positive control inhibitor))*100.

Dose dilution series are prepared for the compounds enabling the testingof dose-response effects in the TYK2 assay and the calculation of theIC₅₀ for each compound. Each compound is routinely tested atconcentration of 20 μM followed by a 1/3 serial dilution, 8 points (20μM-6.67 μM-2.22 μM-740 nM-247 nM-82 nM-27 nM-9 nM) in a finalconcentration of 1% DMSO. When potency of compound series increased,more dilutions are prepared and/or the top concentration is lowered(e.g. 5 μM, 1 μM).

The following compounds have been tested for their activity againstTYK2; and the IC₅₀ values, as determined using the assays describedherein, are given below in Table XVIII.

TABLE XVIII TYK2 IC₅₀ Values of Compounds Cpd # TYK2 IC₅₀ (nM) 1 72.7,73.75, 79.07, 86.77

6.8. TYK2 Kd Determination Assay

TYK2 (Carna Biosciences, 09CBS-0983D) is used at a final concentrationof 5 nM. The binding experiment is performed in 50 mM Hepes pH 7.5,0.01% Brij-35, 10 mM MgCl₂, 1 mM EGTA using 50 nM kinase tracer 236(Invitrogen, PV5592) and 2 nM Eu-anti-GST (Invitrogen, PV5594) withvarying compound concentrations. Detection of tracer is performedaccording to the manufacturers' procedure.

For example, when Compound 1 is tested in this assay, a Kd value of 376nM is measured.

Example 7. Cellular Assays 7.1. JAK-STAT Signalling Assay

HeLa cells are maintained in Dulbecco's Modified Eagle's Medium (DMEM)containing 10% heat inactivated foetal calf serum, 100 U/mL penicillinand 100 μg/mL streptomycin. HeLa cells are used at 70% confluence fortransfection. 20,000 cells in 87 μL cell culture medium are transientlytransfected with 40 ng pSTAT1(2)-luciferase reporter (Panomics), 8 ng ofLacZ reporter as internal control reporter and 52 ng of pBSK using 0.32μL Jet-PEI (Polyplus) as transfection reagent per well in 96-well plateformat. After overnight incubation at 37° C., 10% CO₂, transfectionmedium is removed. 75 μL of DMEM+1.5% heat inactivated fetal calf serumis added. 15 μL compound at 6.7× concentration is added for 60 min andthen 10 μL of human OSM (Peprotech) at 33 ng/mL final concentration.

All compounds are tested in duplicate starting from 20 μM followed by a1/3 serial dilution, 8 doses in total (20 μM-6.6 μM-2.2 μM-740 nM-250nM-82 nM-27 nM-9 nM) in a final concentration of 0.2% DMSO.

After overnight incubation at 37° C., 10% CO₂ cells are lysed in 100 μLlysis buffer/well (PBS, 0.9 mM CaCl₂, 0.5 mM MgCl₂, 5% Trehalose, 0.025%Tergitol NP9, 0.15% BSA).

40 μL of cell lysate is used to read β-galactosidase activity by adding180 μL β-Gal solution (30 μL ONPG 4 mg/mL+150 μL β-Galactosidase buffer(0.06 M Na₂HPO₄, 0.04 M NaH₂PO₄, 1 mM MgCl₂)) for 20 min. The reactionis stopped by addition of 50 μL Na₂CO₃ 1 M. Absorbance is read at 405nm.

Luciferase activity is measured using 40 μL cell lysate plus 40 μL ofSteadylite® as described by the manufacturer (Perkin Elmer), on theEnvision (Perkin Elmer).

10 μM of a pan-JAK inhibitor is used as a positive control (100%inhibition). As negative control 0.5% DMSO (0% inhibition) is used. Thepositive and negative controls are used to calculate z′ and ‘percentinhibition’ (PIN) values.

Percentage inhibition=((fluorescence determined in the presence ofvehicle−fluorescence determined for sample with test compound present)divided by (fluorescence determined in the presence ofvehicle−fluorescence determined for sample without trigger))*100.

PIN values are plotted for compounds tested in dose-response, EC₅₀values are derived and disclosed in Table XIX below

TABLE XIX JAK-STAT EC₅₀ values Cpd # EC₅₀ (nM) 1 922.5, 625.6, 987.7,1767

7.2. OSM/IL-1β Signaling Assay

OSM and IL-1β are shown to synergistically upregulate MMP13 levels inthe human chondrosarcoma cell line SW1353. The cells are seeded in 96well plates at 15,000 cells/well in a volume of 120 μL DMEM (Invitrogen)containing 10% (v/v) FBS and 1% penicillin/streptomycin (InVitrogen)incubated at 37° C. 5% CO₂. Cells are preincubated with 15 μL ofcompound in M199 medium with 2% DMSO 1 hr before triggering with 15 μLOSM and IL-1β to reach 25 ng/mL OSM and 1 ng/mL IL-1β, and MMP13 levelsare measured in conditioned medium 48 h after triggering. MMP13 activityis measured using an antibody capture activity assay. For this purpose,384 well plates (NUNC, 460518, MaxiSorb black) are coated with 35 μL ofa 1.5 μg/mL anti-human MMP13 antibody (R&D Systems, MAB511) solution for24 hrs at 4° C. After washing the wells 2 times with PBS+0.05% Tween,the remaining binding sites are blocked with 100 μL 5% non-fat dry milk(Santa Cruz, sc-2325, Blotto) in PBS for 24 hr at 4° C. Next, the wellsare washed twice with PBS+0.05% Tween and 35 μL of 1/10 dilution ofculture supernatant containing MMP13 in 100-fold diluted blocking bufferis added and incubated for 4 hr at room temperature. Next the wells arewashed twice with PBS+0.05% Tween followed by MMP13 activation byaddition of 35 μL of a 1.5 mM 4-Aminophenylmercuric acetate (APMA)(Sigma, A9563) solution and incubation at 37° C. for 1 hr. The wells arewashed again with PBS+0.05% Tween and 35 μL MMP13 substrate (Biomol,P-126, OmniMMP fluorogenic substrate) is added. After incubation for 24hrs at 37° C. fluorescence of the converted substrate is measured in aPerkin Elmer Wallac EnVision 2102 Multilabel Reader (wavelengthexcitation: 320 nm, wavelength emission: 405 nm).

Percentage inhibition=((fluorescence determined in the presence ofvehicle−fluorescence determined for sample with test compound present)divided by (fluorescence determined in the presence ofvehicle−fluorescence determined for sample without trigger))*100.

For example, when Compound 1 is tested in this assay, an EC₅₀ value of2242.5 (±1098.5) nM is measured.

7.3. PBL Proliferation Assay

Human peripheral blood lymphocytes (PBL) are stimulated with IL-2 andproliferation is measured using a BrdU incorporation assay. The PBL arefirst stimulated for 72 hrs with PHA to induce IL-2 receptor, then theyare fasted for 24 hrs to stop cell proliferation followed by IL-2stimulation for another 72 hrs (including 24 hr BrdU labeling). Cellsare preincubated with test compounds 1 h before IL-2 addition. Cells arecultured in RPMI 1640 containing 10% (v/v) FBS.

Example 8. In Vivo Assays 8.1. PK/PD Study 8.1.1. Dog BioavailabilityStudy 8.1.1.1. Experimental Set Up

The aim of this experiment is to compare the PK in healthy male beagledogs (3 dogs per group) after a single oral administration of Compound 1or as Compound 1.HCl.3H₂O formulated as capsules of two differentstrengths (25 and 100 mg).

The dogs are not fasted before dosing, and have free access to water.Every day of the treatment, a half food ration is provided after the TOblood sampling, 8 to 17 min before treatment, and the second half rationis given just after dosing or 1 h after treatment for period 2. A 3 dayswashout period is ensured between treatments.

Compound 1 or Compound 1.HCl.3H₂O is administered to a target dose of 10mg/kg in capsules (either 4×25 mg, or 1×100 mg capsule). The capsulecomposition is described in Table XX below.

TABLE XX 25 and 100 mg capsules composition Component 25 mg capsule 100mg capsule Compound 1 25.325 mg 101.3 mg Acdisol 4 mg 4 mg Aerosil 1 mg1 mg Avicel 243.1 mg 71 mg Magnesium stearate 1 mg 1 mg

The capsules are administered orally with water (5-10 mL), to providegood oesophageal transit. Each animal is checked at least once daily.

Blood is collected from the jugular vein into lithium heparinised tubesat TO (before food administration) and then at 1 h, 2 h, 4 h, 6 h, 8 h,10 h, and 24 h post treatment.

Plasma is then obtained from blood by centrifugation (2500 g for 10 minat 4° C.), and stored at −20° C. until analysis.

8.1.1.2. Plasma Analysis

Representative aliquots of plasma are diluted with control dog plasma asnecessary to ensure the concentrations present are within the range ofthe calibration curve, and extracted by protein precipitation with 2volumes of acidified (with 0.1% formic acid) acetonitrile containingdeuterated Compound 1 as internal standard (at 150 ng/mL).

After vortex mixing and centrifugation at 4° C., the supernatants arediluted with a 0.5 volume of HPLC grade water in a midi-eppendorf96-well plate. The plate is sealed and shaken to ensure samplehomogeneity prior to analysis. Samples are assayed for Compound 1 byLC-MS/MS using a Waters TQS mass spectrometer, against a series ofmatrix matched calibration and quality control standards.

The Waters TQS method has a standard curve range of 1.00 ng/mL (lowerlimit of quantitation for undiluted samples), to maximally 4000 ng/mLfor Compound 1.

Pharmacokinetic analysis is performed using WinNonlin™ software version5.3, using concentrations from individual animals. Non-compartmentalanalysis is applied to determine the PK parameters (C_(max), T_(max),AUC_(0-last), t_(1/2), etc. . . . )

Concentrations below the limit of detection are set to zero fordescriptive statistics and PK parameter calculations.

The actual doses of Compound 1 administered to each dog are used fordose normalisation of PK parameters (C_(max) and AUC). The results arepresented in Table XXI below and in Error! Reference source not found.

TABLE XXI Dog bioavailability study results Compound form Free baseHCl•trihydrate Dose (single oral 4 × 25 mg/kg 100 mg/kg 4 × 25 mg/kg 100mg/kg administration) Exposure AUC 10.4 11.1 33.6 21.7 (μg · h/mL) Tmax(h) 6.0 8.0 h 2.0 2.0 Cmax (μg/mL) 0.894 0.797 3.05 2.63 T_(1/2) (h)Range 4.43-8.96

Compound 1 (as a free base) is taken orally an therefore passes throughthe HCl containing acidic gastric route, where Compound 1.HCl should beformed. The skilled person would therefore expect to see no differencebetween the two administered forms.

However, as illustrated in Error! Reference source not found., onaverage and at the 2 capsule strengths, Compound 1.HCl.3H₂O is morerapidly absorbed, and shows in vivo improved exposure over Compound 1,which may result in lower dosage regimen, and thereby improved patientcompliance, and potentially lower toxicity, or drug-drug interactionproblems.

FINAL REMARKS

It will be appreciated by those skilled in the art that the foregoingdescriptions are exemplary and explanatory in nature, and intended toillustrate the invention and its preferred embodiments. Through routineexperimentation, an artisan will recognize apparent modifications andvariations that may be made without departing from the spirit of theinvention. All such modifications coming within the scope of theappended claims are intended to be included therein. Thus, the inventionis intended to be defined not by the above description, but by thefollowing claims and their equivalents.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication are specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

It should be understood that factors such as the differential cellpenetration capacity of the various compounds can contribute todiscrepancies between the activity of the compounds in the in vitrobiochemical and cellular assays.

At least some of the chemical names of salt of the invention as givenand set forth in this application, may have been generated on anautomated basis by use of a commercially available chemical namingsoftware program, and have not been independently verified.Representative programs performing this function include the Lexichemnaming tool sold by Open Eye Software, Inc. and the Autonom Softwaretool sold by MDL, Inc. In the instance where the indicated chemical nameand the depicted structure differ, the depicted structure will control.

REFERENCES

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1. (canceled)
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 5. (canceled) 6.(canceled)
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 15. (canceled)16. A pharmaceutically acceptable salt of a compound according toFormula (I):

wherein the salt is a 1:1 free base/maleic acid salt.
 17. Apharmaceutically acceptable salt according to claim 16, wherein the saltis in a crystalline form.
 18. A pharmaceutically acceptable saltaccording to claim 16, wherein the salt exhibits peaks on a XRPDspectrum.
 19. A method for the prophylaxis and/or treatment of acondition selected from inflammatory conditions, autoimmune diseases,proliferative diseases, allergy, transplant rejection, diseasesinvolving degradation and/or disruption of cartilage homeostasis,congenital cartilage malformations, and/or diseases associated withhypersecretion of IL6 or interferons, comprising administering aneffective amount of a pharmaceutically acceptable salt according toclaim
 16. 20. The method according to claim 19, wherein thepharmaceutically acceptable salt is administered in combination withanother therapeutic agent.
 21. The method according to claim 19, whereinthe further therapeutic agent is an agent for the prophylaxis and/ortreatment of inflammatory conditions, autoimmune diseases, proliferativediseases, allergy, transplant rejection, diseases involving degradationand/or disruption of cartilage homeostasis, congenital cartilagemalformations, and/or diseases associated with hypersecretion of IL6 orinterferons.
 22. A method for preparing a pharmaceutically acceptablesalt of claim 16 comprising: combining the compound according to FormulaI with maleic acid in an inert solvent; and precipitating said salt fromsaid solvent.
 23. A method for preparing a pharmaceutically acceptablesalt of claim 16 comprising: combining the compound of Formula I andmaleic acid in a suitable solvent in order to achieve full dissolution;evaporating the solvent in order to achieve supersaturation; andcrystallizing the salt.
 24. The method according to claim 22, whereinthe salt is obtained by mixing the compound of Formula I and the acid ina molar ratio of between 5:1 and 1:5 of Formula I: acid.
 25. The methodaccording to claim 22, wherein the solvent is selected fromacetonitrile, dioxane, THF, acetone, DCM, and MeOH.
 26. Thepharmaceutically acceptable salt according to claim 16, obtainable by:i) suspending 1 equivalent of the compound of Formula I in 20 volumes of5% water in acetone at 25° C., ii) warming the suspension of step i) to50° C., iii) adding 2.1 equivalent of maleic acid (1 M solution in THF)to the stirred suspension at 50° C. obtained in the previous step ii),iv) cooling the mixture of step iii) to 25° C. at 1° C./min and stirringat 25° C. for 22 h, v) separating by filtration the resulting solidobtained in the previous step iv), vi) drying under suction saidresulting solid obtained in the previous step v).